Prevention and treatment of retinal ischemia and edema

ABSTRACT

The present invention relates to methods of treating retinopathy, retinal ischemia and/or retinal edema comprising administering an integrin or integrin subunit antagonist, leukocyte adhesion-inducing cytokine antagonist or growth factor antagonist, a selectin antagonist or adhesion molecule antagonist.

RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.11/651,334, filed Jan. 9, 2007, which is a Continuation of U.S.application Ser. No. 10/364,922, filed Feb. 11, 2003, which is aContinuation of U.S. application Ser. No. 09/474,523 filed Dec. 29,1999, which is a Continuation-in-Part of U.S. application Ser. No.09/248,752, filed Feb. 12, 1999, which claims the benefit of U.S.Provisional Application No. 60/114,221, filed Dec. 30, 1998. The entireteachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant 2P01HL32262-15 from the National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Diabetes affects over 16 million Americans. The World HealthOrganization indicates that diabetes afflicts 120 million peopleworldwide, and estimates that this number will increase to 300 millionby the year 2025. Diabetics are faced with numerous complicationsincluding kidney failure, non-traumatic amputations, an increase in theincidence of heart attack or stroke, nerve damage, and loss of vision.Diabetic retinopathy is a form of visual impairment suffered bydiabetics.

In particular, diabetic retinopathy is responsible for 13.1% and 18.2%newly reported cases of blindness for men and women, respectively.Kohner E. M., et al. Diabetic Retinopathy Metabolism, 25:1985-1102(1975). The prevalence of blind diabetics in the population is about 100people per million. Id.

Less than optimal methods of treatment for diabetic retinopathy exist.For example, laser treatment may be used to slow the progression ofedema, but it cannot be used to reverse the symptoms of diabetes.Accordingly, a need exists to develop effective methods of treatment toreduce or impede vision loss and/or diabetic retinopathy.

SUMMARY OF THE INVENTION

The present invention relates to methods for inhibiting the binding of aleukocyte to an endothelial cell or another leukocyte in the retinalvasculature. The present invention pertains to methods of treating(e.g., reducing or preventing) retinal injury in a mammal (e.g., human,individual, patient) wherein the injury involves retinal edema orretinal ischemia, comprising administering a compound that inhibits thebinding of a leukocyte to endothelium or to another leukocyte wherein areduction in edema or ischemia (e.g., non-perfusion) occurs. Thecompound comprises an integrin antagonist (e.g., lymphocyte functionassociated molecule-1 (LFA-1), Mac-1 or p150,95), a selectin (e.g.,P-selectin, E-selectin and L-selectin) antagonist, an adhesion moleculeantagonist (e.g., Intercellular Adhesion Molecule (ICAM)-1, ICAM-2,ICAM-3, Platelet Endothelial Adhesion Molecule (PCAM), Vascular CellAdhesion Molecule (VCAM)), or a leukocyte adhesion-inducing cytokine orgrowth factor antagonist (e.g., Tumor Neucrosis Factor-α (TNF-α),Interleukin-1 β (IL-1 β), Monocyte Chemotatie Protein-1 (MCP-1) and aVascular Endothelial Growth Factor (VEGF)). The integrin antagonist canbe an integrin subunit (e.g., CD18 or a CD11b) antagonist. Theantagonist can be administered with or without a carrier (e.g.,pharmaceutically acceptable carrier).

In particular, the invention pertains to methods of treating orpreventing retinal injury in a mammal comprising administering to themammal an adhesion molecule antagonist and/or an integrin antagonist,wherein the adhesion molecule antagonist and/or the integrin antagonistinhibits leukocyte interaction, thereby reducing or preventing retinalinjury. The antagonist can be administered in a carrier (e.g., apharmaceutically acceptable carrier). The antagonist for adhesionmolecule can be a VCAM, PCAM, ICAM-2 or ICAM-3 antagonist or,preferably, an ICAM-1 antagonist. In particular, the antagonist can bean antibody or an antibody fragment which is specific for ICAM-1, anantisense molecule that hybridizes to the nucleic acid sequence whichencodes ICAM-1, or a peptide mimetics molecule, a ribozyme, an aptamer,or a small molecule antagonist that inhibits ICAM-1. The integrinantagonist can be a LFA-1 antagonist, Mac-1 antagonist or p150,95antagonist. The integrin antagonist also comprises an integrin subunitantagonist (e.g.,a CD18 antagonist and/or a CD11b antagonist). Theantagonist can be an antibody or antibody fragment specific for CD18and/or CD11b, an antisense molecule that hybridizes to the nucleic acidsequence that encodes CD18 and/or CD11b, or a peptide mimetic molecule,a ribozyme, an aptamer or a small molecule antagonist that inhibits CD18or CD11b.

Another aspect of the invention includes a method for preventing ortreating an individual having retinal injury (e.g., injury caused bydiabetic retinopathy), wherein the injury is associated with retinaledema and/or retinal ischemia, comprising administering to theindividual a compound that inhibits Mac-1 or a pathway thereof Thecompound inhibits ICAM-1, CD18, CD11b, and/or VEGF, and causes adecrease of ischemia and/or edema (e.g., between about 10% and about90%). Leukocyte interaction can also be reduced. The compound can be anantibody, an antibody fragment, a peptide mimetic molecule, an antisensemolecule, a ribozyme, an aptamer and/or a small molecule antagonist.Examples for such a compound are ICAM-1, CD18, CD11b, and/or VEGF.

The invention also pertains to a method of treating an individual havingretinopathy or at risk for retinopathy (e.g., diabetic retinopathy)comprising administering an antagonist (e.g., ICAM-1, CD 18, CD11band/or VEGF), as described herein. The antagonist can optionally beadministered in a suitable carrier (e.g., pharmaceutically acceptablecarrier). Administration of this antagonist results in a decrease inretinal ischemia and/or retinal edema. Preferably, a decrease inischemia and/or edema occurs by at least about 10%, and more preferably,by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% (e.g., between 10%and 95%). Accordingly, the present invention also relates to methods fortreating or preventing retinal edema and/or retinal ischemia comprisingadministering an ICAM antagonist (e.g., ICAM-1), a CD18 antagonist, aCD11b antagonist and/or a VEGF antagonist, wherein a decrease in theedema and/or ischemia occurs.

The present invention also relates to methods of treating diabeticretinopathy by administering an ICAM-1, CD18, CD11b and/or VEGFantagonist and at least one additional antagonist that inhibits thebinding of a leukocyte to an endothelial cell or to another leukocyte.The additional antagonist can be an integrin antagonist (e.g., anintegrin subunit antagonist such as CD18 and/or CD11b), a selectinantagonist, a leukocyte adhesion-inducing or growth factor antagonist,or adhesion molecule antagonist. The additional antagonist can be, forexample, another ICAM antagonist (e.g., an antagonist that is specificfor a different portion or epitope of the ICAM-1 molecule), a PCAMantagonist or a VCAM antagonist. The types of integrin antagonists,selectin antagonists, and leukocyte adhesion-inducing or growth factorantagonists are described herein.

The invention also encompasses a method of inhibiting leukocyteinteraction, comprising contacting a leukocyte, an endothelial cell or aleukocyte adhesion-inducing cytokine, with a compound or antagonist, asdefined herein. The compound can be an integrin antagonist (e.g., anintegrin sub-unit antagonist such as CD18 and/or CD11b), a selectinantagonist, an adhesion molecule antagonist or a leukocyteadhesion-inducing cytokine or growth factor antagonist. In particular,the invention relates to a method of inhibiting leukocyte interaction,comprising contacting an endothelial cell with an adhesion moleculeantagonist (e.g., ICAM-1 specific antagonist), an integrin subunitantagonist (e.g., CD18 and/or CD11b specific antagonist), or a leukocyteadhesion-inducing cytokine antagonist or growth factor antagonist (e.g.,TNF-1α, IL-1β, MCP-1 and VEGF antagonist).

The invention also pertains to a method of preventing or reducingretinal leukostasis an a mammal comprising administering to the mammalan effective amount of an ICAM, CD18, CD11b and/or VEGF antagonist. Thetypes of antagonist is described herein. The method results in retinalleukostasis reduction by between about 10% and 90%.

Another aspect of the invention is a method of decreasing retinalleukocyte adhesion in a mammal, comprising administering to the mammalan effective amount of an antagonist that is specific for CD11b, CD18 ora combination thereof. The retinal leukocyte adhesion is decreasedbetween about 10% and 90%.

Yet another aspect of the invention is a method of treating orpreventing neovascularization in a mammal, comprising administering tothe mammal a CD18 antagonist and an ICAM-1 antagonist, or a CD18antagonist. The types of antagonists are described herein. The method isapplicable to diseases or conditions associated with neovascularizationincluding, but not limited to, age-related macular degeneration,choroidal neovascularization, sickle cell retinopathy, retina veinocclusion, diabetic retinopathy, a condition associated with limbalinjury, a condition associated with increased neovascularization,traumatic alkali injury, Stevens Johnson syndrome and ocular cicatricialpemphagoid. The neovascularization can be reduced in the cornea, theretina or the choroid.

Advantages of the present invention include effective treatment forretinopathy, retinal edema, retinal ischemia, neovascularization andother associated disease. Treatment of these diseases and/or conditionshave been ineffective until the discovery of the present invention. Forthe first time, the present invention provides useful methods oftreatment which target molecules that are involved in these diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a graph showing the density of trapped leukocytes as measuredon 0, 3, 7, 14, 21 and 28 days after diabetes induction. The graph showsa time course of diabetic retinal leukostasis. All data show themean±the standard deviation (SD).

FIG. 1B is a graph showing the retinal vascular [¹²⁵I] albuminpermeation measured 0, 3, 7, 14, 21 and 28 days after diabetesinduction. The graph shows a time course of vascular leakage. All datashow the mean±the standard deviation (SD).

FIGS. 2A-D show four photographs of the same retinal area. FIG. 2A andFIG. 2B show the retinal area after seven days of diabetes. FIG. 2C andFIG. 2D show the retinal area after eight days of diabetes. FIG. 2A andFIG. 2C are photographs from orange leukocyte fluorography (AOLF) andFIG. 2B and FIG. 2D are photographs from fluorescein angiography. Scalebar denotes 100 μm (3.2 pixel=1 μm).

FIG. 3A-F show six photographs of a retinal area. FIGS. 3A and 3B showthe retinal area after one week, FIGS. 3C and 3D show the retinal areaafter two weeks, and FIGS. 3E and 3F show the retinal area after fourweeks. FIGS. 3A, 3C, and 3E are photographs from AOLF and FIGS. 3B, 3Dand 3F are photographs from fluorescein angiography. Scale bar denotes100 μm (3.2 pixel=1 μm).

FIG. 4A is a photograph of ribonuclease protection assay results showingICAM-1 mRNA levels from controls and a diabetic rat three days followingdiabetes induction. Each lane is the signal from the two retinas of asingle animal. The lane labeled “Probes” shows a hundred-fold dilutionof the full-length ICAM-1 and 18S riboprobes. The lanes labeled“RNase-(0.1)” and “RNase-(0.01)” show the ten-fold and hundred-folddilutions, respectively, of the full length riboprobes without sample orRNase.

FIG. 4B is a bar graph showing units of normalized ICAM-1 mRNA forcontrols, three days and seven days after diabetes induction.

FIGS. 5A-D are photographs of the retinal area. The representativeretinal leukostasis is shown in non-diabetic test subjects (FIG. 5A),diabetic test subjects (FIG. 5B), diabetic test subjects given 5 mg/kgmouse control IgG1 (FIG. 5C) and diabetic test subjects treated with 5mg/kg anti-ICAM-1 mAb-treated animals (FIG. 5D). Scale Bars=100 μm; 3.2pixel=1 μm.

FIG. 6A is a bar graph showing the density of trapped leukocytes (×10⁻⁵cells/pixel²) for control, diabetic test subjects not given anything,diabetic test subjects given 5 mg/kg mouse IgG1, diabetic test subjectstreated with 3 mg/kg anti-1CAM-1 antibody, and diabetic test subjectstreated with 5 mg/kg anti-ICAM-1 antibody. NS=Not Significant.

FIG. 6B is a bar graph showing the retinal vascular ¹²⁵I albuminpermeation (μg plasma ×g tissue wet weight^(×1)×min⁻¹) for control,diabetic test subjects not given anything, diabetic test subjects given5 mg/kg mouse IgG1, diabetic test subjects treated with 3 mg/kganti-ICAM-1 antibody, and diabetic test subjects treated with 5 mg/kganti-ICAM-1 antibody. NS=Not Significant.

FIG. 7 is a bar graph showing the amount of adherent neutrophils toendothelium in vitro (thousands per mm²) from control rats and ratshaving Diabetes Mellitus (DM). All data shown are means±StandardDeviation (SD).

FIG. 8 is a bar graph showing the amount of adherent neutrophils(thousands per mm²) for untreated, CD11a, CD11b, CD18, orCD11a/CD11b/CD18 cocktail treated for control and DM rats. All datashown are means±SD.

FIGS. 9A-D are photographs of leukostasis in from AOLF retinas innon-diabetic rat (FIG. 9A), diabetic rat (FIG. 9B), diabetic rat treatedwith the control F(ab′)₂ (FIG. 9C) and anti-CD18 F(ab′)₂ fragmentstreated rats (FIG. 9D).

FIG. 10 is a bar graph showing the density fo trapped leukocytes (×10⁻⁵cells/pixel²) for control, DM, DM and F(ab′)₂, and DM and anti-CD18F(ab′)₂ fragment treated rats.

FIGS. 11A-B are photographs of AOLF retina before (FIG. 11A) and 48hours after a 50 ng Vascular Endothelial Growth Factor (VEGF) injection(FIG. 11B). Scale bar denotes 100 μm (3.2 pixel=1 μm).

FIG. 12 is a bar graph showing the density of trapped leukocytes (×10⁻⁵cells/pixel², mean=SD) in the retina using AOLF for rats injectedintravitreously with 0, 5, 10, 50, 100 ng of VEGF after 48 hours.

FIG. 13 is a bar graph showing the density of trapped leukocytes (×10⁻⁵cells/pixel², mean=SD) in the retina using AOLF for rats injectedintravitreously with the vehicle alone or with 50 ng of VEGF after 6,24, 48, 72, or 120 hours.

FIG. 14 is a bar graph showing the density of trapped leukocytes (×10⁻⁵cells/pixel², mean=SD) in the retina using AOLF for rats injectedintravitreously with the vehicle alone or 50 ng of VEGF with and withoutanti-VEGF mAb treatment after 48 hours.

FIGS. 15A-B are photographs of retina 48 hours after rats were injectedintravitreously with 50 ng (FIG. 15A) followed by fluoresceinangiography (FIG. 15B). Arrows indicate areas of capillary non-perfusiondownstream from static leukocytes. Scale bar denotes 100 μm (3.2 pixel=1μm).

FIGS. 16A-B show VEGF-induced retinal ICAM-1 gen expression. FIG. 16A isa photograph showing results of a ribonuclease protection thatdemonstrated that retinal ICAM-1 levels were significantly increased 20h following the intravitreous delivery of 50 ng VEGF. Control animalsreceived 5 μl of PBS solvent alone. Each lane shows the signal from oneretina of one animal. The lane labeled “Probes” shows a hundred-folddilution of the full-length ICAM-1 and 18S riboprobes. The lanes labeled“RNase-(0.1)” and “RNase-(0.01)” show the ten-fold and hundred-folddilutions, respectively, of the full-length riboprobes without sample orRNase. The lane labeled “RNase+” shows the full-length riboprobes withRNase, but without sample. FIG. 16B is a bar graph showing the amount ofnormalized ICAM-1 mRNA in the retina (arbitrary units, mean+SD) for ratsinjected with the vehicle alone and with 50 ng of VEGF. NS=notsignificant.

FIGS. 17A-B are bar graphs showing the effect of anti-ICAM-1 mAb onpermeability and leukostasis following intravitreous VEGF injection.FIG. 17A is a bar graph showing the retinal vascular [¹²⁵I] albuminpermeation (μg plasma ×g tissue wet weight⁻¹×min⁻¹, mean+SD) for ratsthat were untreated, or treated with the vehicle alone, 50 ng VEGF, 50VEGF and mouse IgG1, or 50 ng VEGF and an anti-ICAM-1 antibody. FIG. 17Bis a bar graph showing the density of trapped leukocytes (×10⁻⁵cells/pixel, mean=SD) in the retina using AOLF for untreated rats orrats treated with treated with the vehicle alone, 50 ng VEGF, 50 VEGFand mouse IgG1, or 50 ng VEGF and an anti-ICAM-1 antibody. ICAM-1bioactivity was inhibited via intravenous administration of ICAM-1neutralizing antibody and retinal permeability (FIG. 17A) or leukostasis(FIG. 17B) were evaluated, respectively. NS=not significant.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods of treating and/or preventing retinalinjury in a mammal by administering to the mammal a compound thatinhibits leukocyte interaction which is the binding of a leukocyte to anendothelial cell or to another leukocyte. Several antagonists inhibitleukocyte interaction and include an integrin antagonist, a selectinantagonist, an adhesion molecule antagonist, or a leukocyteadhesion-inducing cytokine antagonist or growth factor antagonist. Inparticular, an intercellular adhesion molecule-1 antagonist, a CD18antagonist, a CD11b antagonist or a VEGF antagonist are encompassed bythe present method. Administration of such antagonists results in asignificant decrease in retinal edema and/or retinal ischemia. Theretinal injury can be caused by retinopathy or a visually-relateddisease that involves leukocyte occlusion in blood vessels (e.g.,capillaries) and their destruction (e.g., atrophy). In particular, thepresent methods pertain to treating diabetic retinopathy.

Diabetic retinopathy is a progressive degeneration of retinal bloodvessels and is a consequence of diabetes, in particular, diabetesmellitus. One important aspect of the disease is retinal edema. Fluidbuild up from deteriorating blood vessels and capillaries causes edema.As the disease progresses, the damage proliferates and large hemorrhagesand retinal detachment can result.

The term “retinopathy” also refers to noninflammatory degenerativediseases of the retina. The methods of the present invention encompassretinopathy or a visually-related disease that is characterized by oneor more of the following retinal signs: capillary obstruction,nonperfusion, leukostasis, formation of vascular lesions and/orproliferation of new blood vessels in association with ischemic areas ofthe retina. Leukostasis refers to the stasis or non-movement of whiteblood cells (e.g., leukocytes) in the vasculature. Other disorders ordiseases implicated by the invention involve diseases which result inretinal edema and/or retinal ischemia. Examples of such diseases includevein occlusions, sickle cell retinopathy, radiation retinopathy,diabetic retinopathy, VEGF-induced diseases and retinopathy prematurity.

Capillary occlusions constitute a characteristic pathologic feature indiabetic retinopathy, and, when widespread, initiate neovascularization.Neovascularization (e.g., angiogenesis) refers to the formation orgrowth of new blood vessels. Microaneurysms, intraretinal microvascularabnormalities and vasodilation also are commonly found in early stagesof diabetic retinopathy and have been correlated to capillaryocclusions. Schroder, S. et al., American Journal of Pathology, 139(81), 81-100 (1991). Leukocytes cause capillary obstruction that isinvolved in diabetic retinopathy via two mechanisms. This obstruction isthe result of the leukocytes' large cells volume and high cytoplasmicrigidity. Leukocytes can become trapped in capillaries under conditionsof reduced perfusion pressure (e.g., caused by vasoconstriction) or inthe presence of elevated adhesive stress between leukocytes and theendothelium, endothelial swelling, or narrowing of the capillary lumenby perivascular edema. Id. Examples of leukocytes include granulocytes,lymphocytes, monocytes, neutrophils, eosinophils, and basophils.Elevated adhesive stress can result from release of chemotactic factorsor expression of adhesion molecules on leukocytes or endothelial cells.Secondly, leukocytes injures capillaries leading to capillary death,also known as “capillary dropout.”

A number of glycoproteins are involved in the adhesion of leukocytes. Inthe case of neutrophils and monocytes, a family of glycoproteins, knownas β₂ integrins, have been identified. This family of integrins includeLymphocyte Function Associated Antigen-1 (LFA-1), Mac-1, and p150,95.Some integrins are made up of molecules referred to as “subunits” or“integrin subunits.” The LFA-1 integrin is comprised of 2 subunits,CD11a and CD18, Mac-1 integrin is comprised of CD11b and CD18, andp150,95 is made up of CD11c and CD18.

A corresponding family of glycoproteins, referred to as selectins, areexpressed in endothelial cells or can be induced by stimulation withendotoxins or cytokines. The selectins include P-selectin, E-selectin,and L-selectin. The selectin family is involved in endothelialinteraction. Firm adhesion of activated polymorphonuclear neutrophils(PMN) to the endothelial cells occur through the interaction betweenintegrins (e.g., LFA-1, MAC-1 and p150,95) expressed on the PMNs andmembers of the immunoglobulin superfamily of proteins, referred to asIntercellular Adhesion Molecule-1 (ICAM-1), Platelet EndothelialAdhesion Molecule (PCAM), and Vascular Cell Adhesion Molecule (VCAM),expressed by the endothelium. Additionally, cytokines such as TumorNecrosis Factor-α (TNF-α), Interleukin-1 β (IL-1 β), Monocyte ChemotaticProtein-1 (MCP-1), and growth factors (VEGF) can induce the surfaceexpression of ICAM-1, VCAM-1, and E-selectin on endothelial cells.

Intercellular adhesion molecules are involved in and are important forinflammation responses. Mediators of inflammation cause an induction ofICAM-1 expression on various cell types and sites of inflammation. Bothsoluble and membrane forms of ICAM-1 exist. Roep, B. O. et al., Lancet343, 1590-1593, 1590 (1994). ICAM-1 is an inducible cell surface ligandfor LEA-1. Larson, R. S., et al., Immunological Reviews, 114, 181-217,192 (1990). ICAM-1 is a single chain glycoprotein with a peptidebackbone of 55 kD. ICAM-1 is a member of immunoglobulin super familyconsisting of 5 immunoglobulin-like domains. ICAM-1 is expressed orinduced by inflammatory mediators on many cell types includingendothelial cells, epithelial cells, keratinocytes, synovial cells,lymphocytes, and monocytes. The LFA-1 binding site is the firstimmunoglobulin domain of ICAM-1. ICAM-1 also binds with Mac-1, animportant mechanism in retinal edema and retinal ischemia. Various formsof ICAM-1 can be used to generate antagonists, such as antibodies orantisense molecules.

Retinal leukostasis is a very early event in diabetic retinopathy withimportant functional consequences. Both retinal vascular leakage andnon-perfusion follow its development. The inhibition of ICAM-1 activityblocks diabetic retinal leukostasis and potently prevents blood-retinalbarrier breakdown. Leukostasis is associated with the development ofvascular nonperfusion and thus its inhibition can also prevent capillarydropout. Indeed, activated leukocytes are increased in diabetes andleukocytes have been associated with capillary loss in the diabeticchoroid. The data described herein demonstrate that ICAM-1-mediatedleukostasis is increased in the retinal vasculature very early indiabetes and accounts for the majority of diabetes-associated retinalvascular leakage. Thus, these data, described herein, indicate ICAM-1 asa new therapeutic target for the prevention of many of thesight-threatening retinal abnormalities, especially those associatedwith diabetes. See Example 1.

The data described herein also show that CD11a, CD11b, and CD18 β₂integrin levels were increased on the surface of neutrophils fromdiabetic rats. The increases correlated with the enhanced functionaladhesiveness of diabetic neutrophils to rat endothelial cell monolayers.Similarly, in an in vivo model of experimentally-induced diabetes, useof anti-CD18 F(ab′)₂ fragments significantly decreased diabetic retinalleukostasis by 62%, confirming the relevance of the in vitro findings.The data described herein indicate that the Mac-1 integrin complex isoperative in the adhesion of diabetic neutrophils to the retinalcapillary endothelium. Since a major ligand for Mac-1 is ICAM-1, theseresults are consistent with data, shown in Example 1, that ICAM-1blockade prevents diabetic retinal leukostasis and blood-retinal barrierbreakdown, See also Example 2.

Based on the data described herein, it is reasonable to believe that theleukocyte adhesive changes in this model of diabetes are of a systemicnature. The assayed neutrophils were isolated from the peripheral blood,and therefore reflected systemic neutrophil adhesion molecule expressionand bioactivity. The causes of the surface integrin changes remainunknown, however they are likely to be linked to hyperglycemia. Forexample, hyperglycemia directly impacts TNFα expression, a cytokineknown to activate integrin adhesion molecules on leukocytes. In vitrowork has also shown that hyperglycemia promotes increased leukocyteadhesion to endothelium via ICAM-1 and CD18. Thus, hyperglycemia, eitherdirectly or indirectly, is a proximal stimulus for the ICAM-1 and CD18upregulation seen in diabetes.

Also, these data show that a low-level retinal leukostasis occurs in thenormal state. The same molecules that are operative in the diabeticstate also mediate this presumably normal phenomenon. If the low-levelleukostasis in the non-diabetic state is physiologic, then thespecificity of an anti-integrin therapy can be compromised.

The results, described herein, also provide additional evidence ofleukocyte involvement in the pathogenesis of diabetic retinopathy. Theaggregate data indicate that diabetic retinopathy should be, in onesense, redefined as an inflammatory disease. Very early in diabetesleukocytes adhere to the vascular endothelium, trigger breakdown of theblood-retinal barrier, impede flow, and in some instances, extravasateinto the retinal parenchyma. The identification of Mac-1 as a functionaladhesive molecule in diabetic retinopathy provides a target for theprevention and/or treatment of the disease.

Data described herein also show that VEGF induces retinal vascularpermeability and leukostasis through ICAM-1. Retinal leukostasis wasalso spatially linked to capillary non-perfusion. The vitreousconcentration at which VEGF begins to induce these changes (12.5 nM) iswithin the range of vitreous VEGF concentrations observed in human eyeswith diabetic retinopathy. The leukostasis observed in these studies wasspecific to VEGF because co-injection of a neutralizing antibodyabrogated the response. Finally, these findings are consistent with ourdata showing VEGF-induced ICAM-1 expression in the retinal vasculature.See Example 3.

Leukocytes, via their own VEGF, serve to amplify the direct effects ofVEGF when they bind to endothelium. VEGF has been demonstrated inneutrophils, monocytes, eosinophils, lymphocytes and platelets. The factthat some leukocytes possess high affinity VEGF receptors and migrate inresponse to VEGF makes this scenario even more likely.

The data also show that VEGF-induced capillary non-perfusion occursdownstream from areas of leukocyte adhesion. Leukocyte-mediatednon-perfusion characterizes experimental diabetic retinopathy. Indiabetes, patent capillaries become occluded downstream from newlyarrived static leukocytes. Later, following the disappearance of theleukocytes, the capillaries reopen. Since neutrophil and monocytediameters can exceed those of retinal capillary lumens,leukocyte-mediated flow impedance is a likely mechanism.

Taken together, these data indicate that VEGF-induced vascularpermeability is mediated by ICAM-1-mediated retinal leukostasis. Thesedata are the first to show that a non-endothelial cell type contributesto VEGF-induced vascular permeability. They are also the first toprovide a mechanism for the capillary non-perfusion induced by VEGF.Given these findings, targeting ICAM-1 proves useful in the treatment ofdiseases characterized by VEGF-induced vascular changes, such asdiabetic retinopathy.

The invention takes advantage of the surprising discovery thatinhibiting integrins, and in particular the Mac-1 or a pathway thereof,results in a reduction in retinal edema and/or retinal ischemia. Thisreduction in both retinal edema and/or retinal ischemia provides aneffective treatment for various ocular diseases, including retinopathy.In one aspect of the invention, an antagonist's biological activityrefers to a compound that inhibits the Mac-1 integrin adhesion or apathway thereof. Inhibition can occur directly (e.g., by inhibitingbinding of the Mac-1 molecule or a subunit thereof such as CD18 orCD11b), or indirectly (e.g., by inhibiting a molecule that affects Mac-1such as by inhibiting ICAM-1 expression or Vascular Endothelial GrowthFactor (VEGF) expression). The surprising results of directly inhibitingMac-1 by inhibiting Mac-1 subunits are shown in Example 2. Severalmolecules indirectly impact on Mac-1's biological activity (e.g., itsability to bind to ICAM-1, induce leukocyte adhesion, induceleukostasis, cause edema and/or cause ischemia). For example, ICAM-1directly binds to Mac-1. Inhibiting ICAM-1 reduces retinal edema andischemia. See Example 1. Similarly VEGF mediates ICAM-1 expression inthe retinal vasculature, and induces vascular permeability andnon-perfusion. Inhibiting VEGF results in decreased expression ofICAM-1, and a reduction in both retinal edema and retinal ischemia. SeeExample 3. Additionally, TNF-α, a cytokine, induces ICAM-1 expression,which, in turn, can stimulate and increase leukocyte adhesion.Inhibiting the TNF-α pathway, significantly reduces leukocyte adhesion.See Example 2. Inhibiting Mac-1 and molecules that affect the Mac-1pathway (e.g., ICAM-1 expression) unexpectedly results in reductions ofretinal edema and ischemia.

Inhibition of a molecule encompassed by the invention (e.g., Mac-1,CD18, CD11b, ICAM-1, VEGF or TNF-α) can be accomplished in several ways.A molecule can be made inactive or its action disrupted. For example,the expression of these molecules can be inhibited prior to the moleculeexiting the cell using, for example, antisense technology, etc. Themolecule can also be made inactive by inhibiting its binding to areceptor after it exits the cell or is exposed on the membrane of thecell, e.g., with an antibody or antibody fragment. Additionally, theaction of these molecules can be inhibited by disrupting the signalingdownstream from the receptor (e.g. alterations in phosphorylation).These and other methods can be used so long as the activity or action ofone or more of the molecule described herein is inhibited or disrupted.

The invention relates to preventing or treating retinal injury whereinthe retinal injury involves retinal edema and/or retinal ischemia,comprising administering a compound that inhibits the binding of aleukocyte to an endothelial cell or another leukocyte in, for example, ablood vessel or capillary, which results in the reduction of retinaledema and/or retinal ischemia. The term “retinal injury” is definedherein as a decreased ability for the retina to function normally asmeasured, for example, by the patient's vision, electrical signalpotential, fluorescein angiograms or other known methods or methodsdeveloped in the future. The compound has the ability to inhibit orreduce leukocyte occlusion in the retinal vasculature. As describedherein, the compound can be an integrin antagonist (e.g., Mac-1antagonist), an integrin subunit antagonist (e.g., CD18 antagonist or aCD11b antagonist), a selectin antagonist, a leukocyte adhesion-inducingcytokine antagonist or growth factor antagonist (e.g., TNF-α, IL-1β,MCP-1 and VEGF antagonist), or an adhesion molecule antagonist (e.g., anICAM-1, ICAM-2, ICAM-3, PCAM or VCAM antagonist). In particular, theinvention also pertains to administering an ICAM-1 antagonist, a VEGFantagonist, a Mac-1 antagonist, a CD18 antagonist, or a CD11bantagonist, to treat retinal edema, retinal ischemia, and/or diabeticretinopathy. The various forms of the antagonists are described herein.

The methods described herein can be used for treating ocular tissue thatexperiences leukostasis, edema and/or ischemia. Such tissue includes theretina, and the choroid. For example, the invention includes a method oftreating or reducing leukostasis, edema and/or ischemia in the retina orthe choroid of an affected mammal by administering to the mammal one ora combination of any one of the antagonists described herein.

The invention includes methods of inhibiting leukocyte interaction,comprising contacting a leukocyte or endothelial cell with anantagonist. For example, using the various antagonists described herein,one can contact a leukocyte with an integrin antagonist, an endothelialcell with an adhesion molecule antagonist or a selectin antagonist, orsubject the cytokines that induce surface expression of ICAM-1, VCAM-1,and E-selectin to a leukocyte adhesion-inducing cytokine antagonist.

The invention further comprises the use of an ICAM-1, a CD18, a CD11b ora VEGF antagonist in conjunction with a second antagonist. Geneticvariability that exists among various patient populations and/oradditional mechanisms can warrant administering more than oneantagonist. Any combination of the above antagonists can be used. Forexample, the present methods include administering an ICAM-1 antagonist,which is specific to a particular epitope of ICAM-1, and an additionalICAM-1 antagonist, which is specific to a different epitope or geneticvariation. Similarly, an ICAM-1 antagonist can be administered with anyone of the antagonists described herein. Administering a combination ofantagonists to prevent the leukocyte adhesion to endothelial cellsand/or leukocytes results in even more effective treatment of diabeticretinopathy or a more dramatic reduction in retinal edema and/orischemia. See Example 2 in which both a CD18 and CD11b antagonist wasused to reduce leukocyte adhesion. Given the causal effect of leukocyteadhesion on retinal edema and/or ischemia, as proven by the data,administration of a CD18 and/or CD11b antagonist is expected to reduceretinal edema and/or retinal ischemia. The combination of antagonistscan be administered at substantially the same time, or sequentially,with suitable intervals between administration of the antagonists toconfer the desired effect.

The invention also relates to decreasing or reducing the amount ofischemia and/or edema present in an individual by administering aneffective amount or an ICAM-1 a CD18, a CD11b or a VEGF antagonist.Ischemia refers to tissue which lacks proper or suitable blood flow.Ischemia refers to an inadequate circulation of blood flow which can bethe result of a mechanical obstruction (e.g., trapped leukocyte) of theblood supply or damage to the blood supplying vessel which results in areduction of the blood flow. Inadequate blood flow results in reducedtissue oxygenation. Hence, ischemia can be a function of leukostasis,and can be measured by determining the density of trapped leukocytes,and other methods known in the art or developed in the future asdescribed herein.

Edema refers to the build up of excess fluid caused by vasculatureleakage (e.g., vascular permeability). Edema also refers to the build upor accumulation of fluid when the fluid is not timely or properlycleared. As described herein, leukocytes become trapped in thecapillaries in the conditions of reduced perfusion pressure (e.g.,caused by constriction as seen in early stages of diabetes) or in thepresence of an elevated adhesive stress between leukocytes andendothelium, endothelial swelling or narrowing of the capillary lumen byperivascular edema. The leukocyte build up can cause leakage from theblood vessel. Thus, edema can be measured by determining the amount ofretinal vascular albumin permeation, as referred to as “vascularpermeability,” as described herein.

The methods of treatment described herein include reducing or decreasingthe amount of ischemia and/or edema by administering antagonist thatinhibits leukocyte and endothelial cell interaction, as describedherein. The decrease in ischemia is at least about 10% and can begreater, such as at least about 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%,or 95%. The decrease in edema is at least about 10%, and can be greater,such as at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, and preferablyat least about 95%.

The reduction or decrease of the retinal edema and/or retinal ischemiacan be determined, as compared to a control, standard, or baseline. Forexample, a measure of edema or ischemia can be made, in a mammal, priorto administering one of the compounds described herein, and one or moretimes subsequent to administration. A percentage change between two ormore measurements, or a value reflecting the change in the measurementscan be determined. The level of edema and/or ischemia can be quantifiedusing methods known in the art, and a decrease, as compared with acontrol, standard, or baseline, indicates successful treatment. Thequantified amounts of edema and/or ischemia can be compared with asuitable control to determine if the levels are decreased. The sample tobe tested can be compared with levels for the specific individual fromprevious time points (e.g., before having diabetic retinopathy, orduring various phases of treatment for the diabetic retinopathy), orwith levels in normal individuals (e.g., an individual without thedisease) or suitable controls. An individual who is being treated fordiabetic retinopathy can be monitored by determining the levels of edemaand/or ischemia at various time points. Such levels of edema and/orischemia can be determined before treatment, during treatment, and aftertreatment. A decrease in the level of ischemia and/or edema, asdescribed herein, indicates successful treatment. Ischemia and/or edemacan be measured using methods now known or those developed in thefuture. See Kohner E. M., et al. Diabetic Retinopathy Metabolism,25:1985-1102 (1975). For example, ischemia and edema can be measuredusing a fluorescein angiogram or by measuring the vision loss in apatient. Edema can also be assessed by measuring electrical signals orpotential, visualizing the retina using a slit lamp, fluoresceinangiogram, or by using a sensitive isotope dilution method.

Another aspect of the invention includes method for treating orpreventing neovascularization. One of the more difficult problems inophthalmology is treating the ocular surface abnormalities thataccompany limbal cell injury. The limbus is a specialized tissue thatmarks the transition between cornea and conjunctiva. Stem cells residein this area and give rise to the normal corneal epithelium. When thelimbus is sufficiently destroyed, an inflammatory cornealneovascularization ensues and a conjunctiva-like epithelium covers thecornea. The latter lacks the smoothness and cohesion of the normalcorneal epithelium, making it optically inferior and prone to erosions.Corneal neovascularization, and the serum it delivers via leaky vessels,supports the abnormal conjunctiva-like surface that covers the cornea.The selective injury of corneal vessels produced a reversion to a morenormal corneal epithelial phenotype. Huang, A. et al Ophthal. 95:228(1988). Unlike the experimental model, the laser injury of cornealvessels has not seen long-term success in humans. Thus, an effectivetreatment for the corneal neovascularization that follows limbal injuryhas previously remained an elusive goal.

Corneal neovascularization secondary to Timbal injury requires, in part,vascular endothelial growth factor (VEGF). VEGF induces intercellularadhesion molecule-1 (ICAM-1) expression in the vasculature of varioustissues. Further, exogenous VEGF induces the adhesion of leukocytes tothe endothelium of ocular surface vessels, a process that can bepartially blocked with anti-ICAM-1 antibodies. The effect of ICAM-1 andits common ligand the β₂ integrin CD18 was tested, on limbalinjury-associated corneal neovascularization and inflammation in apathophysiologically-relevant model.

Corneal neovascularization leads to vision loss in eyes that haveundergone extensive injury to the limbus. This situation characterizes anumber of conditions, including traumatic alkali injury, Stevens Johnsonsyndrome and ocular cicatricial pemphagoid. Other conditions thatinvolve neovascularization are diseases such as age-related maculardegeneration, choroidal neovascularization, sickle cell retinopathy,retina vein occlusion, diabetic retinopathy, a condition associated withlimbal injury and a condition associated with increasedneovascularization. To date, no treatments have proven effective atpreventing the neovascularization associated with these conditions. Apathophysiologically-relevant mouse model of limbal injury was utilizedto test the role of CD18 and intercellular adhesion molecule-1 (ICAM-1)in the production of corneal neovascularization. The data describedherein show that CD18 and ICAM-1 deficient mice have 39% (m=5, p=0.0054)and 33% (n=5, p=0.013) less neovascularization, respectively, whencompared to strain-specific normal controls. Corneal neutrophil countswere reduced by 66% (n=5, p=0.0019) and 39% (n=5, p=0.0016) in the CD18and ICAM-1 deficient mice, respectively. Taken together, these dataidentify CD-18 and ICAM-1 as important mediators of theinflammation-associated neovascularization that follows limbal injury.CD18 and ICAM-1 also serve as therapeutic targets for the treatment ofthe corneal neovascularization associated with limbal injury.

Hence, another embodiment of the invention includes methods of treatingor preventing ocular (e.g., corneal, retinal or choroid)neovascularization in a mammal (e.g., an individual) by administering tothe mammal a CD18 antagonist and an ICAM-1 antagonist or CD18antagonist. The inhibition of both CD18 and ICAM-1, or CD18, result insignificantly less neovascularization, or as compared to a control, asdefined herein. See Example 4.

Hence, the present methods utilize various forms of antagonists. Anantagonist, as defined herein, means a compound that can inhibit, eitherpartially or fully, the binding of a leukocyte to an endothelial cell orto another leukocyte. An antagonist's biological activity also refers toa compound that can reduce or lessen the interaction between a leukocyteand an endothelial cell, or another leukocyte.

The terms, “antagonist” or “antibody,” include proteins and polypeptidesthat are integrin (e.g., LEA-1, Mac-1 or p150,95) antagonists, integrinsubunit (CD18, CD11a or CD11b) antagonists, adhesion molecule (e.g.,ICAM, PCAM or VCAM) antagonists, selectin (e.g., P-selectin, L-selectinor E-selectin) antagonists, or leukocyte adhesion-inducing cytokineantagonists or growth factor antagonists (e.g. antagonists to TNF-α,IL-1β, MCP-1 or VEGF). These terms also include proteins andpolypeptides that have amino acid sequences analogous to the amino acidsequence of the protein, as described herein, and/or functionalequivalents thereof. These terms also encompass various analogues,homologues, or derivatives thereof. Analogous amino acid sequences aredefined to mean amino acid sequences with sufficient identity to theantagonist's amino acid sequence so as to possess its biologicalactivity. For example, an analogous peptide can be produced with“silent” changes in amino acid sequence wherein one, or more, amino acidresidues differ from the amino acid residues of the protein, yet stillpossess its biological activity. Examples of such differences includeadditions, deletions, or substitutions of residues of the amino acidsequence of the protein or polypeptide. Also encompassed by these terms,are analogous polypeptides that exhibit greater, or lesser, biologicalactivity of the antagonist.

Antagonists also include antibody or antibody fragments, peptidemimetics molecules, antisense molecules, ribozymes, aptamers (nucleicacid molecules), and small molecule antagonists. Soluble forms ofmolecules (e.g., soluble ICAM) can also act as an antagonist because itcan bind to the leukocyte, thereby preventing the membrane bound formfrom binding.

The term “antagonist” and “nucleic acid sequence” include homologues, asdefined herein. The homologous proteins and nucleic acid sequences canbe determined using methods known to those of skill in the art. Initialhomology searches can be performed at NCBI against the GenBank (release87.0), EMBL (release 39.0), and SwissProt (release 30.0) databases usingthe BLAST network service. Altshul, S F, et al, J. Mol. Biol. 215: 403(1990); Altschul, S F., Nucleic Acids Res. 25:3389-3402 (1998), theteachings of both are incorporated herein by reference. Computeranalysis of nucleotide sequences can be performed using the MOTIFS andthe FindPatterns subroutines of the Genetics Computing Group (GCG,version 8.0) software. Protein and/or nucleotide comparisons can also beperformed according to Higgins and Sharp (Higgins, D. G. and P. M.Sharp, “Description of the method used in CLUSTAL,” Gene, 73: 237-244(1988)). Homologous proteins and/or nucleic acid sequences are definedas those molecules with greater than 70% sequences identity and/orsimilarity (e.g., 75%, 80%, 85%, 90%, or 95% homology).

Biologically active derivatives or analogs of the antagonists describedherein also include peptide mimetics. Peptide mimetics can be designedand produced by techniques known to those of skill in the art. (seee.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and 5,654,276, the teachingsof which are incorporated herein by reference). These mimetics can bebased, for example, on the protein's specific amino acid sequence andmaintain the relative position in space of the corresponding amino acidsequence. These peptide mimetics possess biological activity similar tothe biological activity of the corresponding peptide compound, butpossess a “biological advantage” over the corresponding amino acidsequence with respect to one, or more, of the following properties:solubility, stability and susceptibility to hydrolysis and proteolysis.

Methods for preparing peptide mimetics include modifying the N-terminalamino group, the C-terminal carboxyl group, and/or changing one or moreof the amino linkages in the peptide to a non-amino linkage. Two or moresuch modifications can be coupled in one peptide mimetic molecule.Modifications of peptides to produce peptide mimetics are described inU.S. Pat. Nos. 5,643,873 and 5,654,276, the teachings of which areincorporated herein by reference. Other forms of the proteins,polypeptides and antibodies described herein and encompassed by thepresent invention, include those which are “functionally equivalent.”This term, as used herein, refers to any nucleic acid sequence and itsencoded amino acid which mimics the biological activity of the protein,polypeptide or antibody and/or functional domains thereof.

The term, “ICAM-1 antagonist” includes antagonists that directly (e.g.,by inhibiting the ICAM-1 molecules itself) or indirectly inhibit ICAM-1(e.g., by inhibiting a molecules that affects induction of ICAM-1 suchas a VEGF antagonist or a TNF-α antagonist). Such antagonist are thosewhich lead to a reduction in edema and/or ischemia. Antagonists alsoinclude other integrin antagonists (e.g., a LFA-1 or p150,95antagonists), selectin antagonists (e.g., P-selectin, E-selectin orL-selectin antagonist) and other adhesion molecule antagonists (e.g.,ICAM-2, ICAM-3, PCAM or VCAM antagonist) or a leukocyteadhesion-inducing cytokine antagonist or growth factor antagonist (anantagonist for TNF-1α IL-1β, MCP-1 or VEGF).

An ICAM-1 antagonist is also a composition that inhibits the binding ofICAM-1 to a receptor or has the ability to decrease or affect thefunction of ICAM-1. Such antagonists include antibodies to ICAM-1 (e.g.,the IA29 antibody), antisense molecules that hybridize to nucleic acidwhich encodes ICAM-1. ICAM-1 antagonists also include ribozymes,aptimers, or small molecule inhibitors that are specific for ICAM-1 orthe nucleic acid that encodes ICAM-1. Antagonists of ICAM-1 includecompounds which inhibit the binding between LFA-1 or Mac-1 and ICAM-1,or compounds that reduce the biological activity or function of ICAM-1.The biological activity of ICAM-1 refers to the ability to bind to LFA-1or, in particular, to Mac-1, the ability to induce leukocyte adhesion,the ability to cause ischemia and/or the ability to cause edema.

The terms “antibody” or “immunoglobulin” refer to an immunoglobulin orfragment thereof having specificity to a molecule involved inleukocyte-leukocyte interaction or leukocyte-endothelium interaction.Examples of such antibodies include anti-integrin antibodies (e.g.,antibodies specific to LFA-1, Mac-1 or p150,35), anti-integrin subunitantibodies (e.g., antibodies specific to CD18, CD11b or a combinationthereof), anti-selectin antibodies (e.g., antibodies specific toP-selection, E-selection and L-selectin), antibodies to leukocyteadhesion-inducing cytokine antagonists or growth factor antagonists(e.g., TNF-α, IL-1β, MCP-1 and VEGF antibodies), and adhesion moleculeantibodies (e.g., ICAM-1, ICAM-2, ICAM-3, PCAM or VCAM antibodies). Forexample, the terms “ICAM-1 antibody,” or “ICAM-1 immunoglobulin” referto immunoglobulin or fragment thereof having specificity for ICAM-1.

The term, “antibody” is also intended to encompass both polyclonal andmonoclonal antibodies including transgenically produced antibodies. Theterms polyclonal and monoclonal refer to the degree of homogeneity or anantibody preparation and are not intended to be limited to particularmethods of production. An antibody can be raised against an appropriateimmunogen, such as an isolated and/or recombinant polypeptide (e.g.,ICAM-1, CD18, CD11b, VEGF, or TNF-α) or portion thereof (includingsynthetic molecules such as synthetic peptides). In one embodiment,antibodies can be raised against an isolated and/or recombinant antigenor portion thereof (e.g., a peptide) or against a host cell whichexpresses recombinant antigen or a portion thereof. In addition, cellsexpressing recombinant antigen (e.g., ICAM-1, CD18, CD11b, VEGF, orTNF-α), such as transfected cells, can be used as immunogens or in ascreening for an antibody which binds the receptor.

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production, can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256:495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991)).

Following immunization, anti-peptide antisera can be obtained from theimmunized animal, and if desired, polyclonal antibodies can be isolatedfrom the serum. As described herein, purified recombinant proteinsgenerated in E. coli were used to immunize rabbits to generate specificantibodies directed against the antigen. These antibodies recognize therecombinant protein expressed in E. coli. Monoclonal antibodies can alsobe produced by standard techniques which are well known in the art(Kohler and Milstein, Nature 256:495-497 (1975); Kozbar et al.,Immunology Today 4:72 (1983); and Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Generally, ahybridoma is produced by fusing a suitable immortal cell line (e.g., amyeloma cell line such as SP2/0) with antibody producing cells. Theantibody producing cell, preferably those of the spleen or lymph nodes,can be obtained from animals immunized with the antigen of interest. Thefused cells (hybridomas) can be isolated using selective cultureconditions, and cloned by limiting dilution. Cells which produceantibodies with the desired specificity can be selected by a suitableassay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, for example, methods whichselect recombinant antibody from a library, by PCR, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies (see e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani etal., U.S. Pat. No. 5,545,807).

For example, the monoclonal antibody, IA29 can be used as describedherein. The IA29 antibody is specific for ICAM-1, and can be purchasedfrom R and D Systems, Minneapolis, Minn. Similarly, the anti-CD11a,anti-CD18, and the anti-CD11b antibodies utilized in the experimentsdescribed herein are the WT. 1 mAb, 6G2 mAb and the MRC OX-42 mAb,respectively, and can be obtained from Serotee, Inc. (Raleigh, N.C.).

Functional fragments of antibodies, including fragments of chimeric,humanized, primatized, veneered or single chain antibodies, can also beproduced. Functional fragments or portions of the foregoing antibodiesinclude those which are reactive with the antigen (e.g., ICAM-1, CD18,CD11b, VEGF, or TNF-α). For example, antibody fragments capable ofbinding to the antigen or portion thereof, including, but not limitedto, Fv, Fab, Fab′ and F(ab′)₂ fragments are encompassed by theinvention. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′)₂ heavy chainportion can be designed to include DNA sequences encoding the CH₁ domainand hinge region of the heavy chain.

It will be appreciated that the antibody can be modified, for example,by incorporation of or attachment (directly or indirectly (e.g., via alinker)) of a detectable label such as a radioisotope, spin label,antigen (e.g., epitope label such as a FLAG tag) or enzyme label,fluorescent or chemiluminescent group and the like, and such modifiedforms are included within the term “antibody.”

A suitable antagonist is also an antisense molecule that can hybridizeto the nucleic acid which encodes the target polypeptide (e.g., ICAM-1,CD18, CD11b, VEGF, or TNF-α). The hybridization inhibits transcriptionand/or synthesis of the protein. Antisense molecules can hybridize toall, or a portion of the nucleic acid. Producing such antisensemolecules can be done using techniques well-known to those of skill inthe art. For example, antisense molecules or constructs can be madeusing method known in the art. DeMesmaeker, Alain, et al., Acc Chem.Res. 28:366-374 (1995), Setlow, Jane K., Genetic Engineering, 20:143-151(1998); Dietz, U.S. Pat. No. 5,814,500, filed Oct. 31, 1996, entitled,“Delivery Construct for Antisense Nucleic Acids and Method of Use,” theteachings all of which are incorporated by reference in their entirety.In particular, constructing an antisense molecule for an ICAM-1antagonist is described in detail in WO 97/46671, entitled, “EnhancedEfficacy of Liposomal Anti-sense Delivery,” the teachings of which areincorporated by reference in their entirety. Additionally, developing anantisense molecule to inhibit a retinal disorder (e.g., retinopathy) isdescribed in Robinson, G. S., et al., Proc. Natl. Acad. Sci.93:4851-4856 (1996).

Administration and Dosages:

The terms “pharmaceutically acceptable carrier” or a “carrier” refer toany generally acceptable excipient or drug delivery device that isrelatively inert and non-toxic. The antagonist can be administered withor without a carrier. A preferred embodiment is to administer theantagonist (e.g., ICAM-1 antagonist) to the retinal area or thevasculature around or leading to the retina. Exemplary carriers includecalcium carbonate, sucrose, dextrose, mannose, albumin, starch,cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, riceflour, magnesium stearate, and the like. Suitable formulations andadditional carriers are described in Remington's PharmaceuticalSciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of whichare incorporated herein by reference in their entirety. The antagonistcan be administered systemically or locally (e.g., by injection ordiffusion).

Suitable carriers (e.g., pharmaceutical carriers) also include, but arenot limited to sterile water, salt solutions (such as Ringer'ssolution), alcohols, polyethylene glycols, gelatin, carbohydrates suchas lactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc. Such preparations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the active compounds. They canalso be combined where desired with other active substances, e.g.,enzyme inhibitors, to reduce metabolic degradation. A carrier (e.g., apharmaceutically acceptable carrier) is preferred, but not necessary toadminister an antagonist (e.g., an ICAM-1 antagonist).

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-polyoxypropylene blockpolymers, and the like. Ampules are convenient unit dosages.

Preferably, the antagonist is administered locally to the eye, retinalarea, choroid area or associated vasculature. The antagonist can also beadministered to the cornea of the eye. The antagonist diffuses into theeye and contacts the retina or surrounding vasculature (e.g., eye drops,creams or gels).

One or more antagonists described herein can be administered. Whenadministering more than one antagonist, the administration of theantagonists can occur simultaneously or sequentially in time. Theantagonists can be administered before and after one another, or at thesame time. Thus, the term “co-administration” is used herein to meanthat the antagonists will be administered at times to reduceleukostasis, edema and/or ischemia. The methods of the present inventionare not limited to the sequence in which the various antagonists areadministered, so long as the antagonists are administered close enoughin time to produce the desired effect. The methods also includeco-administration with other drugs that used to treat retinopathy orother diseases described herein.

The compositions of the present invention can be administeredintravenously, parenterally, orally, nasally, by inhalation, by implant,by injection, or by suppository. The composition can be administered ina single dose or in more than one dose over a period of time to conferthe desired effect.

The actual effective amounts of drug of the present invention can varyaccording to the specific drug being utilized, the particularcomposition formulated, the mode of administration and the age, weightand condition of the patient, for example. As used herein, an effectiveamount of an ICAM antagonist is an amount of the drug which is capableof reducing the edema and/or ischemia levels. Dosages for a particularpatient can be determined by one of ordinary skill in the art usingconventional considerations, (e.g. by means of an appropriate,conventional pharmacological protocol).

EXEMPLIFICATION Example 1 Prevention of Leukostasis and Vascular LeakageDiabetic Retinopathy via ICAM-1 Inhibition

Diabetic retinopathy is a leading cause of adult vision loss andblindness. Much of the retinal damage that characterizes the diseaseresults from retinal vascular leakage and non-perfusion. This studydemonstrates that diabetic retinal vascular leakage and non-perfusionare temporally and spatially associated with retinal leukocyte stasis(leukostasis) in the rat model of streptozotocin-induced diabetes.Retinal leukostasis increases within days of developing diabetes andcorrelates with the increased expression of retinal intercellularadhesion molecule-1 (ICAM-1). ICAM-1 blockade with a monoclonal antibodyprevents diabetic retinal leukostasis (e.g., resulting in ischemia) andvascular leakage (e.g., resulting in edema) by 48.5% and 85.6%,respectively. These data identify the causal role of leukocytes in thepathogenesis of diabetic retinopathy and demonstrate the importantutility of ICAM-1 inhibition as a therapeutic strategy for theprevention of diabetic retinopathy.

While retinal vascular leakage and non-perfusion are recognized as twomajor complications of diabetes, their pathogenesis remains poorlyunderstood. Leukocytes may be involved in the genesis of thesecomplications. Diabetic retinopathy is not generally considered aninflammatory disease, yet the retinal vasculature of humans and rodentswith diabetes mellitus contains increased numbers of leukocytes. Many ofthese leukocytes are static. The causes and consequences of thisphenomenon are largely unknown. Intercellular adhesion molecule-1(ICAM-1) is a peptide that mediates leukocyte adhesion andtransmigration. ICAM-1 may be operative in the stasis observed indiabetic retinopathy because ICAM-1 immunoreactivity is increased in thediabetic retinal vasculature of humans. However, little is known aboutthe direct pathogenetic role of ICAM-1 in diabetic retinopathy. Thisstudy investigated the mechanisms of diabetic retinal leukocyte stasis(leukostasis) and the role leukocytes play in the development of twosight-threatening complications, vascular leakage and capillarynon-perfusion.

Experimental Procedures

Animals and Experimental Diabetes. Long-Evans rats weighingapproximately 200 g received a single 60 mg/kg injection ofstreptozotocin (Sigma, St. Louis, Mo.) in 10 mM citrate buffer, pH 4.5,after an overnight fast. Control non-diabetic animals received citratebuffer alone. Animals with blood glucose levels greater than 250 mg/dl24 hours later were considered diabetic. Blood pressure was measuredusing a noninvasive cuff sensor and monitoring system (Ueda Electronics,Tokyo, Japan). Blood anticoagulated with EDTA was drawn from theabdominal aorta of each rat after the experiment. The blood sample wasanalyzed using a hematology analyzer. The rats were fed on standardlaboratory chow and were allowed free access to water in anair-conditioned room with a 12-hour light-12-hour dark cycle until theywere used for the experiments.

Acridine Orange Leukocyte Fluorography (AOLF) and FluoresceinAngiography. Leukocyte dynamics in the retina were studied with AOLF(Miyamoto, K., et al., Invest. Ophthalmol. Vis. Sci., 39:2190-2194(1998); Nishiwaki, H., et al., Invest. Ophthalmol. Vis. Sci.,37:1341-1347 (1996); Miyamoto, K., et al., Invest. Ophthalmol. Vis.Sci., 37;2708-2715 (1996)). Intravenous injection of acridine orangecauses leukocytes and endothelial cells to fluoresce through thenon-covalent binding of the molecule to double stranded nucleic acid.When a scanning laser ophthalmoscope is utilized, retinal leukocyteswithin blood vessels can be visualized in vivo. Twenty minutes afteracridine orange injection, static leukocytes in the capillary bed can beobserved. Immediately after observing and recording the staticleukocytes, fluorescein angiography was performed to study therelationship between static leukocytes and retinal vasculature.

Twenty-four hours before AOLF and fluorescein angiography wereperformed, all rats had a heparin-lock catheter surgically implanted inthe right jugular vein for the administration of acridine orange orsodium fluorescein dye. The catheter was subcutaneously externalized tothe back of the neck. The rats were anesthetized for this procedure withxylazine hydrochloride (4 mg/kg) (Phoenix Pharmaceutical, St. Joseph,Mo.) and ketamine hydrochloride (25 mg/kg) (Parke-Davis, Morris Plains,N.J.). Immediately before AOLF, each rat was again anesthetized, and thepupil of the left eye was dilated with 1% tropicamide (Alcon, Humancao,Puerto Rico) to observe leukocyte dynamics. A focused image of theperipapillary fundus of the left eye was obtained with a scanning laserophthalmoscope (SLO; Rodenstock Instrument, Munich, Germany). Acridineorange (Sigma, St. Louis, Mo.) was dissolved in sterile saline (1.0mg/ml) and 3 mg/kg was injected through the jugular vein catheter at arate of 1 ml/min. The fundus was observed with the SLO using the argonblue laser as the illumination source and the standard fluoresceinangiography filter in the 40o field setting for 1 minute. Twenty minuteslater, the fundus was again observed to evaluate leukostasis in theretina. Immediately after evaluating retinal leukostasis, 20 μl of 1%sodium fluorescein dye was injected into the jugular vein catheter. Theimages were recorded on a videotape at the rate of 30 frames/sec. Thevideo recordings were analyzed on a computer equipped with a videodigitizer (Radius, San Jose, Calif.) that digitizes the video image inreal time (30 frames/sec) to 640×480 pixels with an intensity resolutionof 256 steps. For evaluating retinal leukostasis, an observation areaaround the optic disc measuring ten disc diameters in diameter wasdetermined by drawing a polygon surrounded by the adjacent major retinalvessels. The area was measured in pixels and the density of trappedleukocytes was calculated by dividing the number of trapped leukocytes,which were recognized as fluorescent dots, by the area of theobservation region. The densities of leukocytes were calculatedgenerally in eight peripapillary observation areas and an averagedensity was obtained by averaging the eight density values.

Isotope Dilution Technique. Vascular leakage was quantified using anisotope dilution technique based on the injection of bovine serumalbumin (BSA) labeled with two different iodine isotopes, ¹²⁵I and ¹³¹I.Briefly, purified monomer BSA (1 mg) was iodinated with 1 mCi of ¹³¹I or¹²⁵I using the iodogen method. Polyethylene tubing (0.58 mm internaldiameter) was used to cannulate the right jugular vein and the left orright iliac artery. The tubing was filled with heparinized saline. Theright jugular vein cannula was used for tracer injection. The iliacartery cannula was connected to a one ml syringe attached to a HarvardBioscience model PHD 2000 constant-withdrawal pump preset to withdraw ata constant rate of 0.055 ml/min. At time 0, [¹²⁵I]BSA (50 million cpm in0.3 ml of saline) was injected into the jugular vein and the withdrawalpump started. At the eight-minute mark, 0.2 ml (50 million cpm) of[¹³¹I]BSA was injected. At the ten-minute mark, the heart was excised,the withdrawal pump was stopped, and the retina was quickly dissectedand sampled for g-spectrometry. Tissue and arterial samples were weighedand counted in a g-spectrometer (Beckman 5500, Irvine, Calif.). The datawere corrected for background and a quantitative index of [¹²⁵I]BSAtissue clearance was calculated as previously described and expressed asμg plasma×g tissue wet weight-1×min-1. Briefly, [¹²⁵I] BSA tissueactivity was corrected for [¹²⁵I] BSA contained within the tissuevasculature by multiplying [125I]BSA activity in the tissue by the ratioof [¹²⁵I]BSA/[¹³¹I]BSA in the arterial plasma sample obtained at the endof the experiment. The vascular-corrected [¹²⁵I]BSA activity was dividedby the time-averaged [¹²⁵I]BSA plasma activity (obtained from awell-mixed sample of plasma taken from the withdrawal syringe) and bythe tracer circulation time (10 minutes) and then normalized per graintissue wet weight.

Ribonuclease Protection Assay. The retinas were gently dissected freeand cut at the optic disc after enucleation, and frozen immediately inliquid nitrogen. Total RNA was isolated from rat retinas according tothe acid guanidinium thiocyanate-phenol-chloroform extraction method. A425-base pair EcoRI/BamHI fragment of rat ICAM-1 cDNA was prepared byreverse transcription-polymerase chain reaction and cloned intopBluescript II KS vector. A 472 nucleotide antisense riboprobe wasprepared by in vitro transcription (Promega, Madison, Wis.) oflinearized plasmid DNA with T7 RNA polymerase in the presence of[³²P]dUTP. The sequence of the cloned cDNA was verified by DNAsequencing. Twenty micrograms of total cellular RNA were used forribonuclease protection assays. All samples were simultaneouslyhybridized with an 18S riboprobe (Ambion, Austin, Tex.) to normalize forvariations in loading and recovery of RNA. Protected fragments wereseparated on a gel of 5% acrylamide, 8M urea, 1× Tris-borate-EDTA, andquantified with a PhosphorImager (Molecular Dynamics, Sunnyvale,Calif.).

ICAM-1 Blockade. Twenty four hours following streptozotocin injection,confirmed diabetic animals received intraperitoneal injections of 3mg/kg or 5 mg/kg rat ICAM-1 neutralizing antibody (1A29; R&D Systems,Minneapolis Minn.) or 5 mg/kg normal mouse IgG1 (R&D Systems) in sterilephosphate buffered saline. The animals were treated three times perweek. Retinal leukostasis and vascular leakage were studied one weekfollowing diabetes induction.

Statistical Analysis. All results are expressed as means±SD. The datawere compared by analysis of variance (ANOVA) with post-hoc comparisonstested using Fisher's protected least significant difference (PLSD)procedure. Differences were considered statistically significant when Pvalues were less than 0.05.

Results and Discussion

Time-Course Changes of Retinal Leukostasis and Vascular Leakage afterDiabetes Induction. Retinal leukostasis was quantified in Long-Evansrats. Diabetic rats, like humans with diabetes, develop retinalnon-perfusion and increased vascular permeability. FIG. 1 shows the timecourse of diabetic retinal leukostasis and vascular leakage. In FIG. 1A,leukostasis was serially quantified using AOLF. Non-diabetic animals(day 0) and animals with streptozotocin-induced diabetes of varyingduration were studied. Using AOLF, a time course analysis showed thatretinal leukostasis increased 1.9-fold as early as three days followingdiabetes induction (n=5, p<0.05) (FIG. 1A). After one week of diabetes,retinal leukostasis was 3.2-fold higher than in non-diabetic controls(n=5, p<0.0001). This finding remained unchanged in degree for threeadditional weeks (n=5, p<0.0001) (FIG. 1A). Reliable leukostasisquantitation beyond the four-week time point was precluded by cataractformation.

Leukocyte adhesion to endothelial cells can trigger the disorganizationof endothelial cell adherens and tight junctions and vascular leakage.To determine if diabetic retinal leukostasis was correlated withblood-retinal barrier breakdown, retinal albumin permeation wasquantified (FIG. 1B). In FIG. 1B, radioactive albumin permeation intoretinal tissue was quantitated at the same time points using the isotopedilution technique. Retinal albumin permeation characterizes human androdent diabetic retinopathy and can be sensitively quantified using theisotope dilution technique (Tilton, R. G., et al., Diabetes 42:221-232(1993); Tilton, R. G., et al., J. Clin. Invest. 99:2192-2202 (1997); andVinores, S. A., et al., Am. J. Pathol., 134:231-235 (1989). A timecourse analysis in diabetic rats revealed a 2.9-fold (n=8, p<0.0001) and10.7-fold (n=8, p<0.0001) increase in albumin permeation following oneand four weeks of diabetes (FIG. 1B). The breakdown of the blood-retinalbarrier followed the onset of diabetes-associated retinal leukostasis.

Leukocyte-Induced Non-perfusion and Reperfusion in Retinal Capillaries.To further characterize the diabetic retinal leukostasis, serial AOLFand fluorescein angiography studies were performed. FIG. 2 shows thatstatic leukocytes are in flux, block capillary flow and transmigrate.Serial AOLF of static leukocytes in the same retinal area after seven(FIG. 2A) and eight (FIG. 2C) days of diabetes shows their completereplacement within a 24 hour period. The arrow points to a staticleukocyte (FIGS. 2A and 2B) that appears to have transmigrated (FIG.2B). One day later, AOLF and fluorescein angiography show that theleukocyte has disappeared (FIGS. 2C and 2D). The arrowhead shows apatent capillary (FIG. 2B) that subsequently becomes obstructed by astatic leukocyte 24 hours later (FIGS. 2C and 2D). These studiesrevealed that the individual leukocytes observed with AOLF are in flux,even though the overall degree of leukostasis is constant (FIG. 2). Thestatic retinal leukocytes observed seven days following the induction ofdiabetes are topographically distinct from those observed 24 hourslater. Furthermore, a fraction of the leukocytes are in theextravascular space, a result consistent with their rapid transmigrationfollowing dye labeling.

Fluorescein angiography and AOLF were also used to study retinalnon-perfusion. These studies identified static leukocytes directlyassociated with areas of downstream non-perfusion (FIGS. 2 and 3). FIG.3 shows leukocyte-induced non-perfusion and reperfusion. Serial studieswere completed one (FIGS. 3A and 3B), two (FIGS. 3C and 3D) and four(FIGS. 3E and 3F) weeks following diabetes induction using both AOLF(FIGS. 3A, 3C, and 3E) and fluorescein angiography (FIGS. 3B, 3D, and3F). The arrow shows a patent capillary (FIG. 3B) that subsequentlybecomes occluded downstream from a static leukocyte (FIGS. 3C and 3D),and then opens up when the leukocyte disappears (FIGS. 3E and 3F). Thearrowhead shows a patent capillary (FIG. 3B) that becomes occludeddownstream from a static leukocyte (FIGS. 3C and 3D) and then remainsclosed after the leukocyte has disappeared (FIGS. 3E and 3F). Thenon-perfused capillaries were patent prior to the onset of theleukostasis, indicating a causal relationship. As the leukocyte(s)disappeared, the capillaries either reperfused or remained closed (FIG.3). Reperfusion has been observed in human diabetic retinopathy, but themechanism, until now, has remained unexplained.

ICAM-1 Gene Expression in Diabetic Retina. To determine if retinalICAM-1 expression increases in association with diabetic retinalleukostasis, ICAM-1 mRNA levels were quantified using the ribonucleaseprotection assay. FIG. 4 shows ICAM-1 gene expression in diabeticretina. FIG. 4A shows results from a ribonuclease protection assay,which demonstrates that retinal ICAM-1 levels were significantlyincreased seven days following diabetes induction. Each lane shows thesignal from the two retinas of a single animal. The lane labeled“Probes” shows a hundred-fold dilution of the full-length ICAM-1 and 18Sriboprobes. The lanes labeled “RNase-(0.1)” and “RNase-(0.01)” show theten-fold and hundred-fold dilutions, respectively, of the full-lengthriboprobes without sample or RNase. When normalized to 18S RNA, theretinal ICAM-1 levels after seven days of diabetes were 2.2-fold higher(n=4, p<0.05) than in the non-diabetic controls (FIG. 4B). Retinasanalyzed three days following diabetes induction demonstrated thatretinal ICAM-1 mRNA levels were 1.5-fold higher than non-diabeticcontrols, but this increase was not statistically significant (n=5,p>0.05) (FIG. 4). After one week of diabetes, the retinal ICAM-1 levelswere 2.2-fold greater, a significant increase when compared tonon-diabetic controls (n=4, p<0.05). The ICAM-1 increase coincidedtemporally with the development of diabetic retinal leukostasis andblood-retinal barrier breakdown.

An Anti-ICAM-1 Monoclonal Antibody (mAb) Prevents Leukostasis andVascular Leakage in Diabetic Retina. To assess whether ICAM-1 mediatesdiabetic retinal leukostasis, a well characterized ICAM-1 neutralizingantibody (1A29) was used for in vivo adhesion blockade experiments.Tamatani, T. et al., Int. Immunol. 2, 165-171 (1990); Kawasaki, K., etal., J. Immunol. 150, 1074-1083 (1993); Kelly, K. et al., Proc. Natl.Acad. Sci. USA 91, 812-816 (1994). Animals received either 3 or 5 mg/kgintraperitoneal injections of the ICAM-1 antibody three times weekly.Control diabetic animals received an equivalent amount of a non-immuneisotype control antibody. All animals were analyzed one week followingdiabetes induction. The results showed that the ICAM-1 antibody blockeddiabetes-induced leukostasis by 40.9% (3 mg/kg, n=5, p<0.01) and 48.5%(5 mg/kg, n=5, p<0.001) (FIGS. 5 and 6A). The peripheral leukocytecounts at one week increased by 40% (5 mg/kg, n=5, p<0.05) compared tothe control antibody treated animals, a result consistent withsuccessful systemic ICAM-1 blockade (Table 1). Body weight, plasmaglucose and blood pressure were unchanged in all diabetic groups (Table1).

TABLE 1 Characteristics of control, diabetic, mouse IgG1-treateddiabetic, and anti-ICAM-1 mAb-treated diabetic rats Diabetes +Diabetes + 5 mg/kg anti-ICAM-1 mAb Control Diabetes mouse IgG1 3 mg/kg 5mg/kg n 6 CR 7 5 5 5 Body weight (g) 271 ± 12 240 ± 12 * 235 ± 9 * 238 ±6 * 239 ± 12 * Plasma glucose (mg/dl) 123 ± 19 332 ± 35 * 316 ± 61 * 351± 83 * 373 ± 68 * Blood Pressure (mmHg) 111 ± 6 104 ± 12 109 ± 14 105 ±9 105 ± 10 Leukocyte count (×10³/μl)  6.1 ± 6  5.0 ± 1.5 ♦  5.3 ± 0.8 ♦ 6.9 ± 1.4  7.4 ± 2.3 Values are means ± SD. *P < 0.0001 vs. controlrats; ♦P < 0.05 vs. 5 mg/kg anti-ICAM-1 mAb-treated diabetic rats. Allresults are expressed as means ± SD. Unpaired groups of two werecompared using two sample t-test or two sample t-test with Welch'scorrection.To compare three or more groups, analysis of variance was followed bythe post hoc test with Fisher's PLSD procedure. Differences wereconsidered statistically significant when P values were less than 0.05.

The effect of the ICAM-1 inhibition on blood-retinal barrier breakdownwas tested using the same antibody. Animals receiving 3 and 5 mg/kg ofthe anti-ICAM-1 antibody had 63.5% (3 mg/kg, n=4, p<0.0001) and 85.6% (5mg/kg, n=4, p<0.0001) less retinal albumin permeation at one week (FIG.6B), The results suggest that the ICAM-1-dependent component of theleukostasis is largely responsible for the blood-retinal barrierbreakdown.

Example 2 Integrin-Mediated Neutrophil Adhesion and Retinal Leukostasisin Diabetes Introduction:

Leukocyte-endothelial cell interactions in tissues are mediated byadhesion molecules expressed on the surface of leukocytes andendothelial cells. Immunoglobulin superfamily molecules such as ICAM-1are expressed on endothelial cells and bind to β₂-integrins expressed onleukocytes. The integrins are transmembrane receptors that consist ofnoncovalently bound heterodimers composed of α- and β-chains. Theβ₂-integrins are operative in leukocyte adhesion and include LFA-1(lymphocyte function associated antigen, CD11a/CD18), Mac-1 (leukocyteadhesion receptor, CD11b/CD 18) and p150/95 (CD11c/CD18). Each of theβ₂-integrins has a common β-chain in combination with a unique α-chain.CD18 is required for the firm attachment of healthy human neutrophils tohuman umbilical vein endothelial cells.

In vivo studies from our laboratory have investigated the role ofleukocytes in diabetic retinopathy. Utilizing acridine orange leukocytefluorography, the density of static leukocytes in the retinas ofstreptozotocin-induced diabetic rats was demonstrated to be increased.Retinal leukocyte stasis (leukostasis) was observed within three days ofdiabetes induction, and was temporally and spatially correlated withcapillary non-perfusion and blood-retinal barrier breakdown. The onsetof retinal leukostasis coincided with the upregulation of retinal ICAM-1expression. Causality was demonstrated when an anti-ICAM-1 antibodyprevented the diabetes-associated increases in retinal leukostasis andvascular leakage by 48.5% and 85.6%, respectively. However theidentities and bioactivities of the neutrophil adhesion moleculesmediating diabetic retinal leukostasis are less well understood.

The aim of the current study was to investigate in greater detail therole of neutrophils in early diabetic retinal leukostasis. A time pointof one week of diabetes was chosen in this study because steady-stateincreases in diabetic retinal leukostasis and ICAM-1 expression areachieved in one week. Since adhesion can occur in the absence ofincreased adhesion molecule expression, both adhesion moleculeexpression and bioactivity were examined. Finally, the role of CD18 inthe development of diabetic retinal leukostasis was examined in vivousing acridine orange leukocyte fluorography and neutralizing anti-CD 18F(ab′)₂ fragments.

Methods:

Diabetes was induced in Long Evans rats with streptozotocin. Theexpression of the surface integrin subunits CD11a, CD11b, and CD18 onrat neutrophils isolated from peripheral blood was quantitated with flowcytometry. In vitro neutrophil adhesion was studied using quantitativeendothelial cell-neutrophil adhesion assays. The adhesive role of theintegrin subunits CD11a, CD11b and CD18 was tested using specificneutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivowith anti-CD18 F(ab′)₂ fragments and the effect on retinal leukocyteadhesion was quantitated with acridine orange leukocyte fluorography(AOLF).

Animals

Male Long-Evans rats weighing approximately 200 g were used for theseexperiments. The rats were fed standard laboratory chow and allowed freeaccess to water in an air-conditioned room with a 12-hour light-12-hourdark cycle.

Induction of Diabetes

Rats received a single 60 mg/kg intraperitoneal injection ofstreptozotocin (Sigma, St. Louis, Mo.) in 10 mM sodium citrate buffer,pH 4.5, after an overnight fast. Control non-diabetic animals receivedcitrate buffer alone. Animals with blood glucose levels greater than 250mg/dl 24 hours after injection were considered diabetic. All experimentswere performed one week following the induction of diabetes.

Monoclonal Antibodies and F(ab′)2 Fragments

The monoclonal antibodies (mAb) were murine in origin and were used aspurified IgG. For the in vitro studies, mAbs WT.1 (anti-rat CD11a), 6G2(anti-rat CD18), and MRC OX-42 (anti-rat CD11b) were obtained fromSerotec Inc. (Raleigh, N.C.). FITC-conjugated mouse IgG₁ mAb isotypecontrol was obtained from. PharMingen (San Diego, Calif.). Fluoresceinisothiocyanate (FITC)-conjugated goat anti-mouse IgG₁ Ab was obtainedfrom Caltag Laboratories (Burlingame, Calif.). For the in vivo studies,WT.3 anti-rat LFA-1 beta chain (CD18) F(ab′)₂ fragments were obtainedfrom Seikagaku America (Division of Associates of Cape Cod, Inc.,Falmouth, Mass.). Purified mouse anti-human IgG F(ab′)₂ fragments wereobtained from Jackson ImmunoResearch Laboratories Inc. (West Grove,Pa.).

Flow Cytometry

The surface expression of CD11a, CD11b, and CD18 on rat neutrophils wasdetermined using flow cytometry as previously described. Allport J R, etal., J Immunol., 158:4365-4372 (1997). Briefly, whole bloodanticoagulated with EDTA was obtained from the hearts of ratsanesthetized with inhaled isofluorane. Leukocytes were isolated bydextran sedimentation and hypotonic lysis of contaminating erythrocytes.Aliquots of 5×10⁵ cells in 100 μl RPMI 1640 medium (BioWhittaker,Walkersville, Md.) containing 5% fetal bovine serum (RPMI-5%) wereincubated on ice for 10 min. The tubes were centrifuged at 400×g for 5min at 4° C. The cell pellets were resuspended in 100 μl RPMI-5%containing 20 μg/ml primary mAb to CD11a, CD11b, CD18 or isotype controland incubated for 45 min on ice. Primary mAb were detected withFITC-conjugated goat anti-mouse IgG₁ Ab as previously detailed. Thefluorescence of 10⁴ cells was measured on a FACScan (Becton Dickinson,San Jose, Calif.). Neutrophils were manually gated on the basis of theircharacteristic forward and side light scattering properties. The surfaceexpression is presented as the mean channel fluorescence on alogarithmic scale.

Endothelial Cell-Neutrophil Adhesion Assays

Peripheral blood was obtained from rats anesthetized with inhaledisofluorane via heart puncture with a 16-gauge EDTA flushed needle.Neutrophils were isolated from whole blood by density gradientcentrifugation with NIM•2™ (Neutrophil Isolation Media; CardinalAssociates, Santa Fe, N. Mex.) according to the manufacturer'sinstructions. Preparations contained >94% neutrophils as determined byeosin and methylene blue staining (Leukostat staining system; FischerScientific, Pittsburgh, Pa.). There was no red blood cell contamination.The cells were used immediately after collection.

The adhesion of unstimulated neutrophils to confluent monolayers of ratprostate endothelial cells (RPEC) was determined under static conditionsas previously described. (Luscinskas F W, et al., J Immunol., 149:2163(1992); Kiely J M, et al., “Methods in Molecular Biology, AdhesionProtein Protocols,” Leukocyte-endothelial monolayer adhesion assay(static conditions), 131-136 (1999). RPEC were obtained from theAmerican Type Culture Collection (ATCC; Manassas, Va.) and cultured inEagle's minimum essential media (ATCC) supplemented with 5% fetal bovineserum (FBS; GIBCO, Gaithersburg, Md.) and 0.3 ng/ml porcine intestinalheparin (Sigma, St. Louis, Mo.). RPEC were grown to confluence on tissueculture-treated plastic microtiter 96-well plates, stimulated for24-hours with 30 ng/ml recombinant human TNF-α (Genzyme Corp.,Cambridge, Mass.), and incubated for 15 minutes with RPMI-5%. TNF-αstimulation of ICAM-1 surface expression was utilized for allexperiments. Neutrophils were resuspended at 2×10⁶ cells/ml in RPMI-5%and incubated for 10 min at 37° C. with 1 μM of the fluorescent marker,2′,7′-bis-(2-carboxyethyl)-5 (and 6) carboxyfluorescein, acetoxymethylester (Molecular Probes, Eugene, Oreg.) in DMSO (vehicle). Fluorescentlabeled neutrophils were washed once and then incubated in RPMI-5% aloneor RPMI-5% with a saturating concentration of mAb (30 μg/ml) to CD11a,CD11b, or CD18 for 10 min at room temperature. The neutrophils werewashed and then incubated (2×10⁶ neutrophils/ml, 50 μl per well) withRPEC for 10 min at 37° C. Non-adherent cells were removed and thecontent of the wells lysed with 10 mM Tris-HCl, pH 8.4 containing 0.1%SDS. Fluorescence was determined in a microtiter plate fluorimeter(excitation 485 nm, emission 530-540 nm) and the adhesion reported asthe number of adherent neutrophils/mm².

Acridine Orange Leukocyte Fluorography (AOLF)

Leukocyte dynamics in the retina were studied with AOLF, (Miyamoto, K.,et al., “In vivo demonstration of increased leukocyte entrapment inretinal microcirculation of diabetic rats,” Invest Ophthalmol Vis Sci.,39:2190-2194 (1998); Miyamoto, K., et al., Prot Natl Acad Sci USA.,96(19):10836-41 (1999); Nishiwaki, H., et al., Invest Ophthalmol VisSci., 36:123-130 (1995); Nishiwaki, H., et al., Invest Ophthalmol VisSci., 37:1341-1347 (1997)). Rats were anesthetized with 4 mg/kg xylazinehydrochloride (Phoenix Pharmaceutical, St. Joseph, Mo.) and 25 mg/kgketamine hydrochloride (Parke-Davis, Morris Plains, N.J.). The daybefore leukocyte dynamics were observed, a heparin-lock catheter wassurgically implanted in the right jugular vein of each rat. The catheterwas subcutaneously externalized to the back of the neck. Rats receivedintravenous injections of 5 mg/kg anti-rat beta chain (CD18, WT.3)F(ab′)₂ fragments or 5 mg/kg anti-human IgG isotype control F(ab′)₂fragments in sterile phosphate buffered saline 24 hours before AOLF wasperformed. The experiments were carried out in a masked fashion.

Immediately before AOLF, each rat was again anesthetized, and the pupilof the left eye was dilated with 1% tropicamide (Alcon, Humancao, PuertoRico) to observe leukocyte dynamics. A focused image of theperipapillary fundus of the left eye was obtained with a scanning laserophthalmoscope (SLO; Rodenstock Instruments, Munich, Germany). Acridineorange (Sigma, St. Louis, Mo.) was dissolved in sterile saline (1.0mg/ml) and 3 mg/kg was injected through the jugular vein catheter at arate of 1 ml/min. The fundus was observed with the SLO using the argonblue laser as the illumination source and the standard fluoresceinangiography filter in the 40° field setting for 1 min. Twenty min later,the fundus was again observed to evaluate leukostasis in the retina. Theimages were recorded on videotape at the rate of 30 frames/sec. Thevideo recordings were analyzed on a computer equipped with a videodigitizer (Radius, San Jose, Calif.) that digitizes the video image inreal time (30 frames/sec) to 640×480 pixels with an intensity resolutionof 256 steps. For evaluating retinal leukostasis, an observation areaaround the optic disc measuring five disc diameters in radius wasdetermined by drawing a polygon bordered by the adjacent major retinalvessels. The density of trapped leukocytes was calculated by dividingthe number of static leukocytes (recognized as fluorescent dots) by thearea of the observation region (in pixels). The density of staticleukocytes was calculated in 8-10 peripapillary observation areas and anaverage density (×10⁻⁵ cells/pixel²) was obtained.

Blood pressures and heart rates were measured using a noninvasive cuffsensor and monitoring system (Ueda Electronics, Tokyo, Japan). Bloodanticoagulated with EDTA was drawn from the abdominal aorta of each ratafter the experiment to determine the leukocyte count using a hematologyanalyzer. The leukocyte count was determined using a hematologyanalyzer.

Statistical Analysis

All results are expressed as means±SD. The data were compared byanalysis of variance (ANOVA) with post-hoc comparisons tested usingFisher's protected least significant difference (PLSD) procedure.Differences were considered statistically significant when p values wereless than 0.05.

Results

Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n=5,p=0.006), 54% (n=5, p=0.045) and 38% (n=5, p=0.009) greater in diabeticvs. non-diabetic animals, respectively. Seventy-five percent moreneutrophils from diabetic vs. non-diabetic animals adhered to ratendothelial cell monolayers (n=6, p=0.02). Pre-treatment of leukocyteswith either anti-CD11b or anti-CD18 antibodies lowered the proportion ofadherent diabetic neutrophils by 41% (n=6, p=0.01 for each treatment),while anti-CD11a antibodies had no significant effect (n=6, p=0.5). Invivo, systemic administration of anti-CD18 F(ab′)₂ fragments decreaseddiabetic retinal leukostasis by 62% (n=5, p=0.001).

Increased Surface Integrin Expression on Diabetic Neutrophils

Integrin expression was measured on the surface of neutrophils fromnormal and diabetic rats. As shown in Table 2, the flow cytometricanalyses demonstrated statistically significant increases in thediabetic leukocyte CD11a, CD11b, and CD18 levels, as evidenced by theincreases in mean channel fluorescence. Neutrophil CD11a, CD11b, andCD18 levels were 62% (n=5, p=0.006), 54% (n=5, p=0.045), and 38% (n=5,p=0.009) greater, respectively, on the one week-diabetic leukocytes vs.the non-diabetic leukocytes. Integrin expression was similarly increasedon two week-diabetic neutrophils with CD11a, CD11b, and CD18 levelsbeing 53%, 24%, and 38% greater, respectively.

TABLE 2 Flow-cytometric analysis of integrin molecule expression onneutrophils. Diabetes + Diabetes + anti-CD18 Control Diabetes controlF(ab;)₂ F(ab′)₂ n 5 5 5 5 Body Weight 268 ± 10 236 ± 4* 233 ± 7*  237 ±17* (g) Plasma glucose 122 ± 21  327 ± 40* 357 ± 60* 351 ± 28* (mg/dl)Blood pressure 110 ± 7  106 ± 13 103 ± 8  103 ± 7  (mmHg) Leukocyte  6.4± 1.4  4.9 ± 1.6† 5.0 ± 1.3 6.7 ± 0.9 count (×10³ μL) Values are means ±SD. *P < 0.001 vs. control rats †P < 0.05 vs. anti-CD18 F(ab′)²-treateddiabetic rats.

Diabetic neutrophils exhibit increased adhesion to TNFα-activatedendothelial cell monolayers in vitro

The functional adhesion of purified neutrophils to cultured endothelialcell monolayers was investigated. Adhesion assays were performed byadding diabetic or non-diabetic neutrophils to TNF-α-stimulated ratendothelial cell monolayers under static conditions. TNF-α was added tomaximize endothelial cell ICAM-1 expression. Preliminary experimentsdemonstrated a 2.7-fold increase in endothelial cell ICAM-1 expressionwith TNF-α (n=4, p<0.0001). FIG. 7 shows that adhesion of control anddiabetic rat neutrophils to confluent TNF-activated rat endothelial cellmonolayers under static conditions. Neutrophils isolated from diabeticrats demonstrated significantly increased adhesion to rat endothelialcell monolayers. FIG. 7 shows that adhesion of control and diabetic ratneutrophils to confluent TNF-activated rat endothelial cell monolayersunder static conditions. Neutrophils isolated from diabetic ratsdemonstrated significantly increased adhesion to rat endothelial cellmonolayers.

As shown in FIG. 7, 75% more neutrophils from the diabetic rats adheredto the endothelial cell monolayers than neutrophils isolated fromnon-diabetic rats (n=6, p=0.02).

The β₂-integrin molecules mediating neutrophil adhesion in vitro wereexamined. FIG. 8 shows the effect of anti-integrin antibodies onneutrophil adhesion in vitro. Neutrophils were pre-incubated withanti-CD11a, anti-CD11b, anti-CD18 (30 μg/ml of each mAb), or anequimolar mixture of anti-CD11a/CD11b/CD18 antibodies prior to their usein the adhesion studies. In a representative experiment shown in FIG. 8,untreated diabetic neutrophils exhibited increased adhesion toTNFα-activated endothelial cell monolayers under all treatmentconditions. Pretreatment with anti-CD11b or anti-CD18 antibodies eachdecreased diabetic neutrophil adhesion by 41% (n=6, p=0.01 for eachtreatment). In contrast, pretreatment with the anti-CD11a antibody didnot significantly affect diabetic neutrophil adhesion (n=6, p=0.5 vs.untreated diabetic neutrophils). Moreover, treatment with an equimolarmixture of anti-CD11a, anti-CD11b, and anti-CD18 monoclonal antibodiessignificantly reduced diabetic neutrophil adhesion by 72% (n=6, p<0.0001vs. untreated diabetic neutrophils). Non-diabetic neutrophil adhesionwas also reduced with the anti-CD11a, anti-CD11b and anti-CD18antibodies, as well as with the anti-CD11a/CD11b/CD18 antibody cocktail.The decreases were 39%, 49%, 53%, and 52%, respectively (n=6, p<0.05 foreach treatment vs. untreated non-diabetic neutrophils).

In Vivo CD18 Blockade Decreases Leukostasis in Diabetic Rat Retinas

Retinal leukostasis in living animals was measured with AOLF.Intravenous injection of acridine orange causes leukocytes andendothelial cells to fluoresce through the non-covalent binding of themolecule to double stranded DNA. When a scanning laser ophthalmoscope isutilized, retinal leukocytes within blood vessels can be visualized invivo. Twenty minutes after acridine orange injection, static leukocytesin the capillary bed can be observed as fluorescent dots. These labeledcells are leukocytes because blocking CD18, expressed on leukocytes butnot on endothelial cells, causes them to disappear (see below).

Leukocyte dynamics in the retina were observed after CD18 F(ab′)₂blockade as shown in the representative photos of FIG. 9. FIG. 9 showsretinal leukostasis following CD18 blockade. Representative photos fromacridine orange leukocyte fluorography revealed static fluorescentleukocytes in the retinas of control and diabetic rats. The leukostasisin non-diabetic rat retina (FIG. 9A), was increased in diabetic ratretina (FIG. 9B), and unchanged following treatment with the controlF(ab′)₂ (FIG. 9C), however retinal leukostasis was reduced in diabeticrats treated with anti-CD18 F(ab′)₂ fragments (FIG. 9D). As expected,retinal leukostasis was increased in the diabetic vs. non-diabetic ratretinae (FIG. 9B vs. 9A). Treatment of the diabetic rats with theisotype control F(ab′)₂ fragments did not lead to detectable changes inthe degree of leukostasis (FIG. 9C vs. 9B). However, treatment with theanti-CD18 F(ab′)₂ fragments led to a striking decrease in retinalleukostasis (FIG. 9D vs. 9C).

Measurements of leukostasis were obtained throughout the entire retinaeto avoid any potential sampling error and the means and standarddeviations from independent experiments were compared (FIG. 10). FIG. 10shows the quantitation of retinal leukostasis following CD18 blockade.When CD18 bioactivity was inhibited via systemic administration of 5mg/kg of the anti-CD18 neutralizing F(ab′)₂ (clone WT.3), retinalleukostasis was inhibited in diabetic rat retinas. This confirmed thatanti-CD18 blockade significantly decreased leukostasis in diabetic ratsby 62% (n=5, p=0.001 vs. animals receiving control F(ab′)₂) (FIG. 10).The body weight, plasma glucose level, blood pressure, and leukocytecounts for the control and diabetic animals are shown in Table 3. Thediabetic animals all had significantly elevated blood glucose levels anddecreased body weight as compared with the normal rats, as is the norm.Blood pressure was similar among groups. The peripheral leukocyte countsin the diabetic anti-CD18 F(ab′)2-treated animals were increasedcompared to the untreated diabetic animals, a result consistent withsuccessful CD18 blockade.

TABLE 3 Characteristics of control, diabetic, control mAb treateddiabetic, and anti-CD18 F(ab′)₂-treated diabetic rats Control Diabetesp-value n CD11a 115.0 ± 12.8 185.9 ± 18.5 0.006 5 CD11b 182.6 ± 39.2281.9 ± 84.9 0.045 5 CD18 193.2 ± 34.2 267.1 ± 34.3 0.009 5 Values aremeans ± SD of mean channel fluorescence.

The results of the blocking adhesion studies indicate that Mac-1 is thepredominant CD18 integrin involved in diabetic neutrophil adhesion toactivated RPEC monolayers. At present, the reason for a lack of aCD11a-dependent component in diabetic vs. non-diabetic neutrophiladhesion is not known. The residual non-CD18-dependent neutrophiladhesion may be due to the VLA₄-VCAM adhesion pathway because ratneutrophils constitutively express VLA₄ on their surface.

Conclusion: Neutrophils from diabetic animals exhibit higher levels ofsurface integrin expression and integrin-mediated adhesion. In vivo,CD18 blockade significantly decreases leukostasis in the diabeticretinal microvasculature. Integrin adhesion molecules serve astherapeutic targets for the treatment and/or prevention of earlydiabetic retinopathy.

Example 3 Vascular Endothelial Growth Factor (VEGF)-Induced RetinalVascular Permeability is Mediated by ICAM-1 Summary:

Two prominent VEGF-induced retinal effects are vascular permeability andcapillary non-perfusion. The mechanisms by which these effects occur arenot completely known. Using a rat model, it is shown that intravitreousinjections of VEGF precipitate an extensive retinal leukocyte stasis(leukostasis) that coincides with enhanced vascular permeability andcapillary non-perfusion. The leukostasis is accompanied by theupregulation of intercellular adhesion molecule-1 (ICAM-1) expression inthe retina. The inhibition of ICAM-1 bioactivity with a neutralizingantibody prevents the permeability and leukostasis increases by 79% and54%, respectively. These data are the first to demonstrate that anon-endothelial cell type contributes to VEGF-induced vascularpermeability. Additionally, they identify a potential mechanism forVEGF-induced retinal capillary non-perfusion.

In experimental diabetes, the increased presence of static leukocytes inthe retinal circulation is correlated with increased vascularpermeability. The leukostasis and vascular permeability changes coincidewith the upregulation of retinal ICAM-1. When ICAM-1 bioactivity isblocked with an antibody, retinal leukostasis and vascular permeabilityare reduced by 49% and 86%, respectively.

When the retina is bathed in pathophysiologic concentrations of vascularendothelial growth factor (VEGF), enhanced vascular permeability andcapillary non-perfusion are among the vascular changes induced. Themechanisms by which these changes occur are largely unknown. The currentstudies examined the mechanisms underlying VEGF-induced retinalpermeability and non-perfusion. Given the ability of VEGF to increaseICAM-1 expression in the retinal vasculature, the role of ICAM-1 inVEGF-induced vascular permeability and non-perfusion was examined invivo.

Methods:

Animals. Long-Evans rats weighing approximately 200 g were used forthese experiments. They were allowed free access to food and water in anair-conditioned room with a 12-hour light/12-hour dark cycle until theywere used for the experiments.

Intravitreous Injection Procedure. The rats were anesthetized withxylazine hydrochloride (4 mg/kg) (Phoenix Pharmaceutical, St. Joseph,Mo.) and ketamine hydrochloride (25 mg/kg) (Parke-Davis, Morris Plains,N.J.). Intravitreous injections were performed by inserting a 30-gaugeneedle into the vitreous at a site 1 mm posterior to the limbus of theeye. Insertion and infusion were performed and directly viewed throughan operating microscope. Care was taken not to injure the lens or theretina. The head of the needle was positioned over the optic disc, and a5 μl volume was slowly injected into the vitreous. Any eyes thatexhibited damage to the lens or retina were discarded and not used forthe analyses.

Acridine Orange Leukocyte Fluorography (AOLF) and FluoresceinAngiography. Leukocyte dynamics were evaluated using acridine orangeleukocyte fluorography (AOLF). Nishiwaki H, et al., Invest OphthalmolVis Sci, 37:1341-1347 (1996); Miyamoto K, et al., Invest Opthalmol VisSci 39:2190-2194 (1998). Intravenous injection of acridine orange causesleukocytes and endothelial cells to fluoresce through the non-covalentbinding of the molecule to double stranded nucleic acid. When a scanninglaser ophthalmoscope is utilized, retinal leukocytes and blood vesselscan be visualized in vivo. Twenty minutes following acridine orangeinjection, static leukocytes in the capillary bed are observed.

Twenty-four hours before leukocyte dynamics were observed, aheparin-lock catheter was surgically implanted in the right jugular veinfor the administration of acridine orange and sodium fluorescein dye.The catheter was subcutaneously externalized to the back of the neck.The rats were anesthetized for this procedure with xylazinehydrochloride (4 mg/kg) and ketamine hydrochloride (25 mg/kg).

Immediately before AOLF, each rat was again anesthetized, and the pupilof the left eye was dilated with 1% tropicamide (Alcon, Humancao, PuertoRico) to observe leukocyte dynamics. A focused image of theperipapillary fundus of the left eye was obtained with a scanning laserophthalmoscope (SLO; Rodenstock Instrument, Munich, Germany). Acridineorange (Sigma, St. Louis, Mo.) was dissolved in sterile saline (1.0mg/ml) and 3 mg/kg was injected through the jugular vein catheter at arate of 1 ml/min. The fundus was observed with the SLO using the argonblue laser as the illumination source and the standard fluoresceinangiography filter in the 40° field setting for 1 minute. Twenty minuteslater, the fundus was again observed to evaluate retinal leukostasis.The images were recorded on videotape at the rate of 30 frames/sec. Therecordings were analyzed on a computer equipped with a video digitizer(Radius, San Jose, Calif.) that digitizes video images in real time (30frames/sec) at 640×480 pixels with an intensity resolution of 256 steps.For evaluating retinal leukostasis, an observation area around the opticdisc measuring five disc diameters in radius was outlined by drawing apolygon bordered by the adjacent major retinal vessels. The area wasmeasured in pixels and the density of trapped leukocytes was calculatedby dividing the number of static leukocytes, which were recognized asfluorescent dots, by the area of the observation region. The density ofleukocytes was calculated in eight peripapillary observation areas andan average density was obtained by averaging the eight density values.

Immediately after observing and recording the static leukocytes,fluorescein angiography was performed to study the relationship betweenstatic leukocytes and the retinal vasculature. Twenty μl of 1% sodiumfluorescein was injected into the jugular vein catheter and the imageswere captured using the SLO as described above.

Quantitation of Retinal ICAM-1 mRNA Levels. Retinas were gentlydissected free and cut at the optic disc immediately after enucleationand frozen in liquid nitrogen. Total RNA was isolated from rat retinasaccording to the acid guanidinium thiocyanate-phenol-chloroformextraction method. A 425-base pair EcoRI/BamHI fragment of rat ICAM-1cDNA was prepared by reverse transcription-polymerase chain reaction.The PCR product was cloned into pBluescript II KS vector. Afterlinearization by digestion with EcoNI, transcription was performed withT7 RNA polymerase in the presence of [³²P]dUTP generating a 225-basepair riboprobe. An automated DNA sequencer verified the sequence of thecloned cDNA. Ten micrograms of total cellular RNA was used for theribonuclease protection assay. All samples were simultaneouslyhybridized with an 18S riboprobe (Ambion, Austin, Tex.) to normalize forvariations in loading and recovery of RNA. Protected fragments wereseparated on a gel of 5% acrylamide, 8M urea, 1× Tris-borate-EDTA, andquantified with a PhosphorImager (Molecular Dynamics, Sunnyvale,Calif.).

Quantitation of Retinal Vascular Permeability. Vascular leakage wasquantified using the isotope dilution technique. Tilton R G, et al., JClin Invest 99:2192-2202 (1997). Briefly, purified monomer bovine serumalbumin (BSA; Sigma, St. Louis, Mo.) (1 mg) was iodinated with 1 mCi of¹³¹I or ¹²⁵I using the iodogen method. Polyethylene tubing (0.58 mminternal diameter) was used to cannulate the right jugular vein and theleft or right iliac artery. The tubing was filled with heparinizedsaline (400 U heparin/ml). The right jugular vein cannula was used fortracer injection. The iliac artery cannula was connected to a one mlsyringe attached to a Harvard Bioscience model PHD 2000 constantwithdrawal pump preset to withdraw at a constant rate of 0.055 ml/min.At time 0, [¹²⁵I] albumin (50 million cpm in 0.3 ml saline) was injectedinto the jugular vein and the withdrawal pump started. At the eightminute mark, 0.2 ml (50 million cpm in 0.3 ml saline) of [¹³¹I]BSA wasinjected into the jugular vein. At the ten-minute mark, the heart wasexcised, the withdrawal pump was stopped, and the retina was quicklydissected and sampled for γ-spectrometry. Tissue and arterial sampleswere weighed and counted in a γ-spectrometer (Beckman 5500, Irvine,Calif.). The data were corrected for background and a quantitative indexof [¹²⁵I] tissue clearance was calculated as previously described andexpressed as μg plasma ×g tissue wet weight⁻¹×min⁻¹. Briefly, [¹²⁵I]BSAtissue activity was corrected for [¹²⁵I]BSA contained within the tissuevasculature by multiplying [¹²⁵I]BSA activity in the tissue by the ratioof [¹²⁵I]BSA/[¹³¹I]BSA in an arterial plasma sample. Thevascular-corrected [¹²⁵I]BSA activity was divided by the time-averaged[¹²⁵I]BSA plasma activity (obtained from a well-mixed sample of plasmataken from the withdrawal syringe) and by the tracer circulation time(10 min) and then normalized per gram tissue wet weight.

Anti-ICAM-1 Antibody Inhibition of Retinal Vascular Permeability andLeukostasis. To study the in vivo effect of ICAM-1 blockade onVEGF-induced retinal vascular permeability and leukostasis, a wellcharacterized rat ICAM-1 neutralizing monoclonal antibody (mAb) was usedutilized (IA29; R&D Systems, Minneapolis, Minn.). Tamatani T, et al.,Int Immunol, 165-171 (1990); Kawasaki K, et al. J Immunol, 150:1074-1083(1993); Kelly K J, et al., Proc. Natl Acad Sci USA, 91:812-816 (1994).The animals were randomly divided into five groups. The first groupreceived no treatment. The second group received 5 μl ofphosphate-buffered saline (PBS) injected into the vitreous of the lefteye. The third group received 50 ng VEGF₁₆₅ in 5 μl PBS injected intothe vitreous of the left eye (12.5 nM final concentration). The fourthgroup received 50 ng VEGF in PBS injected into the vitreous of the lefteye plus 5 mg/kg isotype-matched normal mouse IgG1 (R&D Systems) givenintravenously. The fifth group received 50 ng VEGF in PBS injected intothe vitreous of the left eye plus 5 mg/kg of the anti-ICAM-1 mAb givenintravenously. Twenty-four hours later, retinal leukocyte dynamics andvascular permeability were quantified.

Statistical Analysis. All results are expressed as the mean±SD. Unpairedgroups of two were compared using the two sample t-test or the twosample t-test with Welch's correction. To compare three or more groups,analysis of variance (ANOVA) followed by the post hoc test with Fisher'sprotected least significant difference (PLSD) procedure was used.Differences were considered statistically significant when P values wereless than 0.05.

Results:

VEGF-induced Retinal. Leukostasis. FIG. 11A shows AOLF appearance of anormal retinal prior to injection of 50 ng VEGF. FIG. 11B shows AOLFappearance of the same retinal area 48 h following intravitreous VEGFinjection. Numerous static leukocytes are visible, as well as vesseldilation and tortuosity. A single 50 ng intravitreous injection ofVEGF₁₆₅ (R& D Systems, Minneapolis, Minn.) in 5 μl PBS was able toinduce marked retinal leukostasis 48 h later (FIG. 11). Vessel dilationand tortuosity were also evident. A dose-response study demonstratedthat a 2.6-fold increase in leukostasis could be induced with as littleas 10 ng VEGF (2.5 nM) (FIG. 12, n=5, p<0.05). A plateau was reachedwith 50-100 ng VEGF (˜4-5-fold, n=5, p=<0.001 to 0.0001). Based on thesedata, the 50 ng dose was chosen for the time course experiments.Intravitreous injections of 50 ng VEGF were followed by AOLF 6, 24, 48,72, and 120 h later. Twenty-four hours following intravitreousinjection, VEGF increased retinal leukostasis 4.8-fold (FIG. 13, n=5,p<0.01 vs. vehicle control). The VEGF-induced leukostasis increasespeaked 48 h post-injection and persisted for at least 120 h (n=5,p<0.01).

To confirm that this effect was due to VEGF alone, four rats received amixture of VEGF with a 50:1 molar excess of a previously characterizedVEGF neutralizing monoclonal antibody (A4.6.1, Genentech, South SanFrancisco, Calif.) (FIG. 14). Co-injection of the anti-VEGF antibodycompletely abrogated the VEGF-induced leukostasis 48 h later (n=4,p<0.001).

VEGF-induced Retinal Capillary Perfusion. FIG. 15 showsleukocyte-induced capillary non-perfusion. FIG. 15A shows the retina 48hours after fifty ng VEGF was delivered via intravitreous injection asmeasured with AOLF. AOLF was immediately followed by fluoresceinangiography and FIG. 15B shows areas of capillary non-perfusiondownstream from static leukocytes. Fluorescein angiography performed 20minutes following AOLF revealed relatively large areas of downstreamcapillary non-perfusion associated with some of the static leukocytes(FIG. 15). The majority of the leukocytes observed appeared to be in theintravascular space. Normal and vehicle injected eyes did not exhibitnon-perfusion.

VEGF-induced Retinal ICAM-1 Gene Expression. Twenty hours followingintravitreous injection of 50 ng VEGF or PBS vehicle alone, total RNAwas isolated from each rat retina and ICAM-1 gene expression wasquantitated using the ribonuclease protection assay (FIG. 16A). Whennormalized to 18S, retinal ICAM-1 levels in the VEGF-injected eyes were2.8-fold greater than in the eyes injected with vehicle alone (FIG. 16B,n=5, p<0.02).

ICAM-1 Blockade of VEGF-induced Vascular Permeability and Leukostasis.Animals receiving intravitreous VEGF had a 3.2-fold increase in vascularpermeability 24 h following injection (FIG. 17A, n=4, p<0.0001 vs.vehicle control). Similarly, there was a 4.3-fold increase in retinalleukostasis (FIG. 17B, n=5, p<0.0001 vs. vehicle control). Intravenoustreatment with the non-immune control antibody did not significantlyalter the degree of VEGF-induced permeability (FIG. 17A, n=3, p>0.05) orleukostasis (FIG. 17B, n=4, p>0.05). However, the animals receivingintravenous anti-ICAM mAb had a 79% reduction in VEGF-induced retinalvascular permeability (FIG. 17A, n=4, p<0.0001 vs. untreated) and a 54%reduction in VEGF-induced retinal leukostasis (FIG. 17B, n=4, p<0.01 vs.untreated).

Example 4 CD18 and ICAM-1 Dependent Corneal Neovascularization andInflammation Following Limbal Injury Materials and Methods. CornealNeovascularization Model

Male CD 18-deficient and ICAM-1-deficient mice were used (Jackson Labs,Bar Harbor, Me.) and strain-specific normal male C57BL/6 mice served ascontrols. The mice were anesthetized with 50 mg/kg intraperitonealpentobarbital sodium and a drop of proparicaine was instilled into theleft eye. A number 15 Bard-Parker blade (vendor and city) was used todebride the corneal epithelium. Two microliters of 0.15 M NaOH was thenapplied topically and the limbal epithelium was removed with a TookeCorneal Knife, 2.5×15 mm Dissecting Blade (Arista, N.Y.) A rotary motionparallel to limbus was utilized. Erythromycin ophthalmic ointment wasinstilled postoperatively.

Measurement of Corneal Neovascularization

For measurement of neovascularization, mice were injected approximately8 μg of the endothelial cell-specific marker BS-1 lectin conjugated toFITC (Vector Laboratory) per 1 g of body weight on day 7 after scrapingor day 2 after implantation of VEGF. In 30 minutes after the injectionof the dye, the eyes were harvested and fixed with 10% neutral bufferedformalin, and then the cornea was flat-mounted on slide glasses.Fluorescence in the flat-mounted cornea was captured using CCD cameraattached to a Leica Fluorescence microscope and saved to Macintosh 6500(Apple computer) as a .tif image file. The images were taken with thesame settings including exposure time on both study group and controlone. The digital images were processed using OpenLab Software andintegrated optical density in the images was measured.

Peripheral Leukocyte Counts

Peripheral blood samples were collected from tail vessels into Eppendorftube with EDTA when cornea that was served for confirming infiltrationof PMN was enucleated. For total leukocyte count blood was incubatedwith Turk solution and then counted manually using Hemocytemeter. Thepreparation of a thin, air-dried edge smear was made to perform themicroscopic manual differential and stained with Giemsa solution. PMNcount was then calculated from the differential.

Corneal Leukocyte Counts

To determine the counts of PMN infiltration in cornea the eyes wereenucleated on day 2 after scraping or implantation of VEGF, and storedin 10% neutral buffered formalin. The tissue was embedded in paraffin,and 5-μm-thick sections were cut and then transferred to slide glasses.The tissue sections were stained with Giemsa stain. The slides were thenobserved microscopically, and the number of PMNs was counted in 5 fields(2 of periphery, 2 of midperiphery and 1 of center) in the cornea frominflammatory models and in 1 field between VEGF pellet and corneallimbus in the cornea from VEGF-induced Neovascularization models.

Statistical Analysis

Student t-test and ANOVA were used for the comparison. Probability lessthan 0.05 was considered significant.

Results: Corneal Neovascularization in CD18 KO, ICAM-1 KO and NormalMice

To determine if CD18 and ICAM-1 were important in the development of thecorneal neovascularization associated with limbal injury, timbal injurywas followed by quantitation of corneal neovascularization 7 d later.Compared to the strain-specific controls, the CD18 null mice 39% fewervessels (n=5, p=0.0054). Similarly, the ICAM-1 null mice has 33% lessneovascularization that the control mice (n=5, p=0.013).

Corneal PMN Density in CD18 KO, ICAM-1 KO and Normal Mice

To determine if the inhibition of corneal neovascularization wasassociated with the decreased transmigration of PMN into the cornea,corneal PMN counts were performed 2 d following limbal injury. This timepoint was chosen because it manifested maximum corneal opacity andcorneal leukocyte infiltration. Compared to the strain-specificcontrols, the CD null mice had 66% fewer PMN (n=5, p=0.0016). The ICAM-1null mice had 65% fewer PMN (n=5, p=0.0019) compared to thestrain-specific controls.

Peripheral Blood PMN Counts in CD18 KO, ICAM-1 KO and Normal Mice

To determine if peripheral PMN cell counts were altered in the animals,standard PMN counts were calculated from the differential. The averagecount in the C57BL6/J controls was 6263±2313.18 vs. counts of 9315±1486and 10,794±2199 in the CD18 and ICAM knockout mice, respectively.

Discussion:

The data indicate that CD18 and ICAM-1 amplify the cornealneovascularization that occurs following limbal injury. The process isassociated with higher corneal leukocyte counts, and the latter arelikely causal, in part, for the increased neovascularization. The dataalso indicate that the CD18 and ICAM-1 KO mice have a higher proportionof circulating leukocytes, a result consistent with absence of CD18 andICAM-1 systemically. It also confirms that the corneal leukocytes likelytransmigrated, and are not the result of systemic leukocytopenia. Takentogether, these data identify CD18 and ICAM-1 mediators of theinflammatory corneal neovascularization in a clinically relevant modelof limbal injury.

Experiments described herein show that limbal injury upregulates VEGF.When VEGF is inhibited, corneal neovascularization is reduced. VEGF isknown to act directly on the endothelial cells and the vasculature,resulting in neovascularization. However, leukocytes augment thisprocess. The mechanism involves VEGF. Leukocytes, via their own VEGF,serve to amplify the direct effects of non-leukocyte VEGF on thevasculature. VEGF has been demonstrated in neutrophils, monocytes,eosinophils, lymphocytes and platelets. It has also been identified inthe neutrophils and monocytes that infiltrate the cornea followinglimbal injury. The fact that some leukocytes possess high affinity VEGFreceptors and migrate in response to VEGF is consistent with thisscenario. Endogenous VEGF triggers leukocyte adhesion, transmigrationand further VEGF release, producing a positive feedback loop.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

1. A method for reducing or preventing retinal injury in a mammal,comprising administering to the mammal a vascular endothelial growthfactor (VEGF) antagonist, wherein said VEGF antagonist inhibitsleukocyte interaction, thereby reducing or preventing retinal injury. 2.The method of claim 1, wherein the antagonist is an antibody or antibodyfragment specific for VEGF.
 3. The method of claim 1, wherein theantagonist is an antisense molecule that hybridizes to the nucleic acidsequence that encodes VEGF, or a peptide mimetic molecule, ribozyme, anaptamer or small molecule antagonist that inhibits VEGF.
 4. The methodof claim 1, wherein the VEGF antagonist is administered in apharmaceutically acceptable carrier.
 5. The method of claim 1, wherein adecrease of at least one condition selected from the group consisting ofretinal edema and retinal ischemia occurs.
 6. The method of claim 5wherein said decrease is between about 10% and about 90%.
 7. The methodof claim 1, wherein the mammal is a human and wherein said human hasdiabetic retinopathy.
 8. A method for treating an individual havingdiabetic retinopathy comprising administering to said individual a VEGFantagonist, wherein leukocyte interaction is reduced or inhibited. 9.The method of claim 8, wherein at least one additional antagonist thatinhibits the binding of a leukocyte to an endothelial cell or to anotherleukocyte is administered to said individual.
 10. The method of claim 9,wherein the additional antagonist is at least one antagonist selectedfrom the group consisting of: an integrin antagonist, selectinantagonist, leukocyte adhesion-inducing cytokine antagonist or anothergrowth factor antagonist and an adhesion molecule antagonist.
 11. Themethod of claim 10, wherein the integrin is selected from a groupconsisting of: LFA-1, Mac-1 and p150,95.
 12. The method of claim 11,wherein the integrin antagonist comprises an integrin subunitantagonist.
 13. The method of claim 12, wherein the integrin subunitantagonist comprises a CD18 antagonist, CD11a antagonist or CD11bantagonist.
 14. The method of claim 10, wherein the selectin is selectedfrom a group consisting of: P-selectin, E-selectin and L-selectin. 15.The method of claim 10, wherein the leukocyte adhesion-inducing cytokineantagonist or growth factor antagonist is selected from a groupconsisting of: TNF-1α, IL-1β, MCP-1 and another VEGF antagonist.
 16. Themethod of claim 10, wherein the adhesion molecule antagonist is selectedfrom the group consisting of: a PCAM antagonist, a VCAM antagonist, anICAM-1 antagonist, an ICAM-2 antagonist and an ICAM-3 antagonist.
 17. Amethod for treating an individual with at least one condition selectedfrom the group consisting of retinal edema and retinal ischemiacomprising administering to the individual a VEGF antagonist, wherein adecrease in retinal edema, retinal ischemia or retinal edema and retinalischemia occurs.
 18. The method of claim 17, wherein said decrease isbetween about 10% and about 90%.